Compositions and Methods Based on PMT Engineering for Producing Tobacco Plants and Products Having Altered Alkaloid Levels

ABSTRACT

The present disclosure provides compositions and methods related to tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants.

INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the file named “P34620US02_SL.txt” whichis 200,704 bytes (measured in MS-Windows®) and created on Jul. 24, 2019,is filed electronically herewith and incorporated by reference in itsentirety.

FIELD

The present disclosure provides tobacco genetic engineering formodulating alkaloid and nicotine levels.

BACKGROUND

Nicotine is the predominant alkaloid, usually accounting for more than90-95% of the total alkaloids in commercial tobacco cultivars. Theremaining alkaloid fraction is primarily comprised three additionalalkaloids: nornicotine, anabasine, and anatabine. Tobacco plants withreduced nicotine levels have been achieved with varying and inconsistentresults by modulating different nicotine biosynthetic genes andtranscriptional regulators. There is a need for new technologies toreduce nicotine levels in tobacco leaves.

SUMMARY

The present disclosure provides tobacco plants with altered totalalkaloid and nicotine levels and commercially acceptable leaf grade,their development via breeding or transgenic approaches, and productionof tobacco products from these tobacco plants.

In an aspect, the present disclosure provides a tobacco plant, or partthereof, comprising one or more mutant alleles in at least one PMT geneselected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, andPMT4, wherein the tobacco plant is capable of producing a leafcomprising a nicotine level less than the nicotine level of a leaf froma control tobacco plant not having the one or more mutant alleles whengrown and processed under comparable conditions.

In another aspect, a tobacco plant comprises one or more mutant allelesin at least two PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4.

In a further aspect, a tobacco plant comprises one or more mutantalleles in at least three PMT genes selected from the group consistingof PMT1a, PMT1b, PMT2, PMT3, and PMT4.

In another aspect, a tobacco plant comprises one or more mutant allelesin at least four PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4.

In a further aspect, a tobacco plant comprises one or more mutantalleles in five PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4.

In an aspect, the present disclosure provides a tobacco plant selectedfrom the group consisting of a single pmt mutant, a double pmt mutant, atriple mutant, a quadruple mutant, and a quintuple mutant, as listed inTables 8A to 8E.

In an aspect, the present disclosure provides a tobacco plant as listedin Tables 4A to 4E or Table 10. In another aspect, the presentdisclosure provides a progeny plant of a tobacco plant in Tables 4A to4E or Table 10, from either selfing or a cross with another plant inTables 4A to 4E or Table 10.

In another aspect, the present disclosure provides a tobacco plantcomprising various combinations of the pmt mutant alleles listed inTables 5A to 5E or Tables 12A to 12E to give rise to a single pintmutant, a double pint mutant, a triple mutant, a quadruple mutant, or aquintuple mutant. In an aspect, the present disclosure provides atobacco plant comprising a pint mutant allele sequence selected from thegroup consisting of SEQ ID Nos. 21 to 200, 410 to 441, 474 to 505, 538to 569, 602 to 633, and 666 to 697.

The present disclosure further provides cured tobacco, tobacco blends,tobacco products comprising plant material from tobacco plants, lines,varieties or hybrids disclosed.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID Nos: 1 to 5 set forth exemplary genomic sequences of PMT1b,PMT1a, PMT2, PMT3 and PMT4, respectfully, from a TN90 reference genome.

SEQ ID Nos: 6 to 10 set forth exemplary cDNA sequences of PMT1b, PMT1a,PMT2, PMT3 and PMT4, respectfully, from TN90.

SEQ ID Nos: 11 to 15 set forth exemplary polypeptide sequences of PMT1b,PMT1a, PMT2, PMT3 and PMT4, respectfully, from TN90.

SEQ ID Nos: 16 to 22 set forth exemplary guide RNA sequences.

SEQ ID Nos: 23 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633,and 666 to 697 set forth exemplary edited pmt mutant sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RNA expression of five PMT genes in TN90 roots

FIG. 2: Nicotine levels in various low-alkaloid lines: CS15 (a quintuplepmt knock-out mutant line CS15 in the NLM (Ph Ph) background), a PMTRNAi transgenic line in the VA359 background) and a low-nicotine KY171(“LN KY171”) variety (the KY 171 background harboring nic1 and nic2double mutations), in comparison to their respective normal-alkaloidcontrol line: NLM (Ph Ph), VA359, and KY171 background.

FIG. 3: Total alkaloid levels in various low-alkaloid lines: CS15, PMTRNAi, and LN KY171, in comparison to their respective normal-alkaloidcontrol line: NLM (Ph Ph), VA359, and KY171 background.

FIG. 4: Leaf yield in various low-alkaloid lines: CS15, PMT RNAi, and LNKY171, in comparison to their respective normal-alkaloid control line:NLM (Ph Ph), VA359, and KY171 background.

FIG. 5: Leaf quality in various low-alkaloid lines: CS15, PMT RNAi, andLN KY171, in comparison to their respective normal-alkaloid controlline: NLM (Ph Ph), VA359, and KY171 background.

Photographs depicting mold growth on cured tobacco, including TN90 LC(FIG. 6A), LA BU 21 (FIG. 6B), TN90 comprising an RNAi construct todownregulate PR50 (FIG. 6C), TN90 comprising an RNAi construct todownregulate PMT genes (FIG. 6D), and TN90 comprising edits to all fivePMT genes (FIG. 6E).

FIG. 7: Depiction of mold infection observed in the lines examined inFIGS. 6A-6E.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. One skilled in the art will recognize many methods can be usedin the practice of the present disclosure. Indeed, the presentdisclosure is in no way limited to the methods and materials described.For purposes of the present disclosure, the following terms are definedbelow.

Any references cited herein, including, e.g., all patents andpublications are incorporated by reference in their entirety.

As used herein, the singular form “a,” “an,’ and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

When the term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth by 10%.

As used herein, phrases such as “less than”, “more than”, “at least”,“at most”, “approximately”, “below”, “above”, and “about”, when used inconjunction with a series of numerical values, modify each and everyvalue within the series. For example, an expression of “less than 1%,2%, or 3%” is equivalent to “less than 1%, less than 2%, or less than3%.”

As used herein, a tobacco plant refers to a plant from the speciesNicotiana tabacum.

Nicotine biosynthesis in tobacco starts with the methylation of thepolyamine, putrescine, to N-methylputrescine by the enzyme, putrescineN-methyltransferase (PMT), using S-adenosyl-methionine as the co-factor.This is a step that commits precursor metabolites to nicotinebiosynthesis. PMT enzymes are classified under the enzyme classificationsystem as EC 2.1.1.53. In Nicotiana tabacum, five genes encodeputrescine N-methyltransferases, designated PMT1a, PMT1b, PMT2, PMT3,and PMT4. Table 1A lists genomic DNA sequences, cDNA sequences, andprotein sequences of these five PMT genes in a TN90 plant. The presentdisclosure describes compositions and methods that are used to edit PMTgenes to produce pmt mutant plants having reduced nicotine levels whilemaintaining leaf quality.

As used herein, “PMT1b” or the “PMT1b gene” refers to a genic locus intobacco encoding a polypeptide having an exemplary amino acid sequencein TN90 as set forth in SEQ ID No. 11.

As used herein, “PMT1a” or the “PMT1a gene” refers to a genic locus intobacco encoding a polypeptide having an exemplary amino acid sequencein TN90 as set forth in SEQ ID No. 12.

As used herein, “PMT2” or the “PMT2 gene” refers to a genic locus intobacco encoding a polypeptide having an exemplary amino acid sequencein TN90 as set forth in SEQ ID No. 13.

As used herein, “PMT3” or the “PMT3 gene” refers to a genic locus intobacco encoding a polypeptide having an exemplary amino acid sequencein TN90 as set forth in SEQ ID No. 14.

As used herein, “PMT4” or the “PMT4 gene” refers to a genic locus intobacco encoding a polypeptide having an exemplary amino acid sequencein TN90 as set forth in SEQ ID No. 15.

As used herein, a mutation refers to an inheritable genetic modificationintroduced into a gene to reduce, inhibit, or eliminate the expressionor activity of a product encoded by the gene. Such a modification can bein any sequence region of a gene, for example, in a promoter, 5′ UTR,exon, intron, 3′ UTR, or terminator region. In an aspect, mutations arenot natural polymorphisms that exist in a particular tobacco variety orcultivar. As used herein, a “mutant allele” refers to an allele from alocus where the allele comprises a mutation.

As used herein, a “pint mutant” refers to a tobacco plant comprising oneor more mutations in one or more PMT genes. A pmt mutant can be a singlemutant, a double mutant, a triple mutant, a quadruple mutant, or aquintuple mutant. As used herein, a single, double, triple, quadruple,or quintuple pmt mutant refers to a mutant having modifications in one,two, three, four, or five PMT genes, respectively. A pmt mutant can alsobe a homozygous mutant, a heterozygous mutant, or a heteroallelic mutantcombination in one or more PMT genes.

As used herein, a gene name or a genic locus name is capitalized andshown in italic, e.g., PMT1a, PMT1b, PMT2, PMT3, and PMT4. A protein orpolypeptide name is capitalized without being italicized, e.g., PMT1a,PMT1b, PMT2, PMT3, and PMT4. A mutant name (for either referencing to ageneral mutation in a gene or a group of genes, or referencing to aspecific mutant allele) is shown in lower case and italic, e.g., pmt,pmt1a, pmt1b, pmt2, pmt3, and pmt4.

In an aspect, the present disclosure provides a tobacco plant, or partthereof, comprising one or more mutant alleles in at least one PMT geneselected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, andPMT4, wherein the tobacco plant is capable of producing a leafcomprising a nicotine level less than the nicotine level of a leaf froma control tobacco plant not having the one or more mutant alleles whengrown and processed under comparable conditions. In an aspect, a singlepmt mutant tobacco plant is provided. In another aspect, a single pmtmutant tobacco plant comprises nicotine at a level below 1%, below 2%,below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%,below 90%, or below 95% of the nicotine level of a control plant nothaving the single pmt mutation when grown in similar growth conditions.In a further aspect, a single pmt mutant tobacco plant comprisesnicotine at a level between 1% and 5%, between 5% and 10%, between 10%and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%,between 50% and 60%, between 60% and 70%, between 70% and 80%, between80% and 90%, or between 90% and 95% of the nicotine level of a controlplant not having the single pmt mutation when grown in similar growthconditions.

In another aspect, a tobacco plant comprises one or more mutant allelesin at least two PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4. In an aspect, a double pmt mutant tobaccoplant is provided. In another aspect, a double pmt mutant tobacco plantcomprises nicotine at a level below 1%, below 2%, below 5%, below 8%,below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95%of the nicotine level of a control plant not having the double pmtmutations when grown in similar growth conditions. In a further aspect,a double pmt mutant tobacco plant comprises nicotine at a level between1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%,between 30% and 40%, between 40% and 50%, between 50% and 60%, between60% and 70%, between 70% and 80%, between 80% and 90%, or between 90%and 95% of the nicotine level of a control plant not having the doublepmt mutations when grown in similar growth conditions.

In a further aspect, a tobacco plant comprises one or more mutantalleles in at least three PMT genes selected from the group consistingof PMT1a, PMT1b, PMT2, PMT3, and PMT4. In an aspect, a triple pmt mutanttobacco plant is provided. In another aspect, a triple pmt mutanttobacco plant comprises nicotine at a level below 1%, below 2%, below5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%,below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below90%, or below 95% of the nicotine level of a control plant not havingthe triple pmt mutations when grown in similar growth conditions. In afurther aspect, a triple pmt mutant tobacco plant comprises nicotine ata level between 1% and 5%, between 5% and 10%, between 10% and 20%,between 20% and 30%, between 30% and 40%, between 40% and 50%, between50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and90%, or between 90% and 95% of the nicotine level of a control plant nothaving the triple pmt mutations when grown in similar growth conditions.

In another aspect, a tobacco plant comprises one or more mutant allelesin at least four PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4. In an aspect, a quadruple pmt mutanttobacco plant is provided. In another aspect, a quadruple pmt mutanttobacco plant comprises nicotine at a level below 1%, below 2%, below5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%,below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below90%, or below 95% of the nicotine level of a control plant not havingthe quadruple pmt mutations when grown in similar growth conditions. Ina further aspect, a quadruple pmt mutant tobacco plant comprisesnicotine at a level between 1% and 5%, between 5% and 10%, between 10%and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%,between 50% and 60%, between 60% and 70%, between 70% and 80%, between80% and 90%, or between 90% and 95% of the nicotine level of a controlplant not having the quadruple pmt mutations when grown in similargrowth conditions.

In a further aspect, a tobacco plant comprises one or more mutantalleles in five PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4. In an aspect, a quintuple pmt mutanttobacco plant is provided. In another aspect, a quintuple pmt mutanttobacco plant comprises nicotine at a level below 1%, below 2%, below5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%,below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below90%, or below 95% of the nicotine level of a control plant not havingthe quintuple pmt mutations when grown in similar growth conditions. Ina further aspect, a quintuple pmt mutant tobacco plant comprisesnicotine at a level between 1% and 5%, between 5% and 10%, between 10%and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%,between 50% and 60%, between 60% and 70%, between 70% and 80%, between80% and 90%, or between 90% and 95% of the nicotine level of a controlplant not having the quintuple pmt mutations when grown in similargrowth conditions.

In an aspect, a tobacco plant is a single pmt mutant, a double pmtmutant, a triple mutant, a quadruple mutant, or a quintuple mutant aslisted in Tables 8A to 8E. In another aspect, a tobacco plant comprisesone or more pmt mutant alleles listed in Tables 5A to 5E and Tables 12Ato 12E. Each and every combination of the pmt mutant alleles listed inTables 5A to 5E and Tables 12A to 12E is also provided to give rise to asingle pmt mutant, a double pmt mutant, a triple mutant, a quadruplemutant, or a quintuple mutant. Each of the mutated loci can be eitherhomozygous or heterozygous, or comprises a heteroallelic combination. Inanother aspect, a tobacco plant comprises a pmt mutant genotypecombination as shown for each individual line listed in Tables 4A to 4Eand Table 10. In an aspect, a tobacco plant comprises a pmt mutantallele sequence selected from the group consisting of SEQ ID Nos. 21 to200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697. Inanother aspect, the present disclosure provides a double pmt mutant, atriple mutant, a quadruple mutant, or a quintuple mutant comprising pmtmutant allele sequences selected from the group consisting of SEQ IDNos. 21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666to 697.

In an aspect, a tobacco plant is capable of producing a leaf comprisinga nicotine level less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the nicotinelevel of a leaf from a control tobacco plant when grown and processedunder comparable conditions. In another aspect, a tobacco plant iscapable of producing a leaf comprising a nicotine level between 1% and5%, between 5% and 10%, between 10% and 20%, between 20% and 30%,between 30% and 40%, between 40% and 50%, between 50% and 60%, between60% and 70%, between 70% and 80%, between 80% and 90%, or between 90%and 95% of the nicotine level of a control tobacco plant when grown andprocessed under comparable conditions.

In another aspect, a tobacco plant is capable of producing a leafcomprising a total alkaloid level less than 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or0.25% of the total alkaloid level of a leaf from a control tobacco plantwhen grown and processed under comparable conditions. In another aspect,a tobacco plant is capable of producing a leaf comprising a totalalkaloid level between 1% and 5%, between 5% and 10%, between 10% and20%, between 20% and 30%, between 30% and 40%, between 40% and 50%,between 50% and 60%, between 60% and 70%, between 70% and 80%, between80% and 90%, or between 90% and 95% of the total alkaloid level of acontrol tobacco plant when grown and processed under comparableconditions.

In a further aspect, a tobacco plant is capable of producing a leafcomprising a total alkaloid level less than 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% of the total alkaloid level of a leaf from a controltobacco plant when grown and processed under comparable conditions.

In an aspect, a mutant pmt allele comprises a mutation in a PMT sequenceregion selected from the group consisting of a promoter, 5′ UTR, firstexon, first intron, second exon, second intron, third exon, thirdintron, fourth exon, fourth intron, fifth exon, fifth intron, sixthexon, sixth intron, seventh exon, seventh intron, eighth exon, 3′ UTR,terminator, and any combination thereof. In another aspect, a mutant pmtallele comprises a mutation in a PMT genomic sequence region listed inTables 1D to 1H.

In another aspect, a mutant pmt allele comprises one or more mutationtypes selected from the group consisting of a nonsense mutation, amissense mutation, a frameshift mutation, a splice-site mutation, andany combination thereof. In an aspect, a mutant pmt allele is a nullallele or a knock-out allele.

In an aspect, a mutant pmt allele results in one or more of thefollowing: a PMT protein truncation, a non-translatable PMT genetranscript, a non-functional PMT protein, a premature stop codon in aPMT gene, and any combination thereof.

In another aspect, a mutant pint allele comprises a mutation selectedfrom the group consisting of a substitution, a deletion, an insertion, aduplication, and an inversion of one or more nucleotides relative to awild-type PMT gene.

In an aspect, a pmt mutant comprises a zygosity status selected from thegroup consisting of homozygous, heterozygous, and heteroallelic. Inanother aspect, a pint mutant is homozygous or heteroallelic in at least1, 2, 3, 4, or 5 PMT genes. In an aspect, a pmt mutant is homozygous orheteroallelic in at least 4 PMT genes. In another aspect, a pint mutantis homozygous or heteroallelic in all five PMT genes. In another aspect,a pint mutant comprises mutations in PMT1a and PMT3.

In an aspect, a tobacco plant is capable of producing a leaf comprisinga nicotine level selected from the group consisting of less than 0.15%,less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, lessthan 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and lessthan 0.01% dry weight.

In another aspect, a tobacco plant is capable of producing a leafcomprising a total alkaloid level selected from the group consisting ofless than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than0.5%, less than 0.4%, less than 0.3%, and less than 0.2% dry weight.

In a further aspect, a tobacco plant is capable of producing a curedleaf comprising a total TSNA level of between 2 and 0.05, between 1.9and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6 and0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and 0.05,between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05,between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05,between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05,between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, orbetween 0.1 and 0.05 ppm.

In an aspect, a tobacco plant is capable of producing leaves, whencured, having a USDA grade index value selected from the groupconsisting of 50 or more, 55 or more, 60 or more, 65 or more, 70 ormore, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. Inanother aspect, a tobacco plant is capable of producing leaves, whencured, having a USDA grade index value comparable to that of a controlplant when grown and cured in similar conditions, where the controlplant shares an essentially identical genetic background with thetobacco plant except for the modification. In a further aspect, atobacco plant is capable of producing leaves, when cured, having a USDAgrade index value of at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 98% of the USDA grade index valueof a control plant when grown in similar conditions, where the controlplant shares an essentially identical genetic background with thetobacco plant except the modification. In a further aspect, a tobaccoplant is capable of producing leaves, when cured, having a USDA gradeindex value of between 65% and 130%, between 70% and 130%, between 75%and 130%, between 80% and 130%, between 85% and 130%, between 90% and130%, between 95% and 130%, between 100% and 130%, between 105% and130%, between 110% and 130%, between 115% and 130%, or between 120% and130% of the USDA grade index value of a control plant. In a furtheraspect, a tobacco plant is capable of producing leaves, when cured,having a USDA grade index value of between 70% and 125%, between 75% and120%, between 80% and 115%, between 85% and 110%, or between 90% and100% of the USDA grade index value of a control plant.

In an aspect, a tobacco plant comprises nicotine at a level below 1%,below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%,or below 80% of the nicotine level of a control plant when grown insimilar growth conditions, where the control plant shares an essentiallyidentical genetic background with the tobacco plant except for themodification.

In a further aspect, a tobacco plant comprises one or more pmt mutantalleles and further comprises a transgene or mutation directlysuppressing the expression or activity of one or more genes encoding aproduct selected from the group consisting of MPO, QPT, BBL, A622,aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase,ornithine decarboxylase, arginine decarboxylase, nicotine uptakepermease (NUP), and MATE transporter.

In an aspect, a tobacco plant comprises one or more pmt mutant allelesand further comprises a mutation in an ERF gene of Nic2 locus. In anaspect, a tobacco plant further comprises one or more mutations in twoor more, three or more, four or more, five or more, six or more, or allseven genes selected from the group consisting of ERF189, ERF115,ERF221, ERF104, ERF179, ERF17, and ERF168. See Shoji et al., Plant Cell,(10):3390-409 (2010); and Kajikawa et al., Plant physiol. 2017,174:999-1011. In an aspect, a tobacco plant further comprises one ormore mutations in ERF189, ERF115, or both.

In an aspect, a tobacco plant comprises one or more qpt mutant allelesand further comprises a mutation in an ERF gene of Nic1 locus (or Nic1blocus as in PCT/US2019/013345 filed on Jan. 11, 2019, published asWO/2019/140297). See also WO/2018/237107. In an aspect, a tobacco plantfurther comprises one or more mutations in two or more, three or more,four or more, five or more, six or more, or seven or more genes selectedfrom the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19,ERF130, ERF16, ERF29, ERF210, and ERF91L2. See Kajikawa et al., Plantphysiol. 2017, 174:999-1011. In an aspect, a tobacco plant furthercomprises one or more mutations in one or more, two or more, three ormore, four or more, five or more, or all six genes selected from thegroup consisting of ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L2.

In an aspect, the present disclosure further provides a pmt mutanttobacco plant, or part thereof, comprising a nicotine or total alkaloidlevel selected from the group consisting of less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%,less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, lessthan 0.025%, less than 0.01%, and less than 0.005%, where the tobaccoplant is capable of producing leaves, when cured, having a USDA gradeindex value of 50 or more 55 or more, 60 or more, 65 or more, 70 ormore, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. Inanother aspect, such pmt mutant tobacco plant comprises a nicotine levelof less than 0.02% and are capable of producing leaves, when cured,having a USDA grade index value of 70 or more. In a further aspect, suchtobacco plant comprises a nicotine level of less than 0.01% and arecapable of producing leaves, when cured, having a USDA grade index valueof 70 or more.

In an aspect, the present disclosure also provides a tobacco plant, orpart thereof, comprising a non-transgenic mutation, where thenon-transgenic mutation reduces the nicotine or total alkaloid level ofthe tobacco plant to below 1%, below 2%, below 5%, below 8%, below 10%,below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below50%, below 60%, below 70%, or below 80% of the nicotine level of acontrol plant when grown in similar growth conditions, where the tobaccoplant is capable of producing leaves, when cured, having a USDA gradeindex value comparable to the USDA grade index value of the controlplant, and where the control plant shares an essentially identicalgenetic background with the tobacco plant except the non-transgenicmutation.

In an aspect, a tobacco plant comprises a pint mutation introduced by anapproach selected from the group consisting of random mutagenesis andtargeted mutagenesis. In another aspect, a pmt mutation is introduced bya targeted mutagenesis approach selected from the group consisting ofmeganuclease, zinc finger nuclease, TALEN, and CRISPR.

Unless specified otherwise, measurements of alkaloid or nicotine levels(or another leaf chemistry or property characterization) or leaf gradeindex values mentioned herein for a tobacco plant, variety, cultivar, orline refer to average measurements, including, for example, an averageof multiple leaves of a single plant or an average measurement from apopulation of tobacco plants from a single variety, cultivar, or line.

Unless specified otherwise, the nicotine or alkaloid level (or anotherleaf chemistry or property characterization) of a tobacco plant ismeasured after topping in a pooled leaf sample collected from leafnumber 3, 4, and 5 after topping. As used herein, whenever a comparisonbetween leaves from two plants (e.g., a mutant plant versus a controlplant) is mentioned, leaves from the same or comparable stalkposition(s) and developmental stage(s) are intended so that thecomparison can demonstrate effects due to genotype differences, not fromother factors. As an illustration, leaf 3 of a wild-type control plantis intended as a reference point for comparing with leaf 3 of a pmtmutant plant. In an aspect, a tobacco plant comprising at least one pmtmutation is compared to a control tobacco plant of the same tobaccovariety.

Nicotine or alkaloid level (or another leaf chemistry or propertycharacterization) of a tobacco plant can also be measured in alternativeways. In an aspect, the nicotine or alkaloid level (or another leafchemistry or property characterization) of a tobacco plant is measuredafter topping in a leaf having the highest level of nicotine or alkaloid(or another leaf chemistry or property characterization). In an aspect,the nicotine or alkaloid level of a tobacco plant is measured aftertopping in leaf number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Inanother aspect, the nicotine or alkaloid level (or another leafchemistry or property characterization) of a tobacco plant is measuredafter topping in a pool of two or more leaves with consecutive leafnumbers selected from the group consisting of leaf number 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, and 30. In another aspect, the nicotine or alkaloidlevel (or another leaf chemistry or property characterization) of atobacco plant is measured after topping in a leaf with a leaf numberselected from the group consisting of between 1 and 5, between 6 and 10,between 11 and 15, between 16 and 20, between 21 and 25, and between 26and 30. In another aspect, the nicotine or alkaloid level (or anotherleaf chemistry or property characterization) of a tobacco plant ismeasured after topping in a pool of two or more leaves with leaf numbersselected from the group consisting of between 1 and 5, between 6 and 10,between 11 and 15, between 16 and 20, between 21 and 25, and between 26and 30. In another aspect, the nicotine or alkaloid level (or anotherleaf chemistry or property characterization) of a tobacco plant ismeasured after topping in a pool of three or more leaves with leafnumbers selected from the group consisting of between 1 and 5, between 6and 10, between 11 and 15, between 16 and 20, between 21 and 25, andbetween 26 and 30.

As used herein, leaf numbering is based on the leaf position on atobacco stalk with leaf number 1 being the youngest leaf (at the top)after topping and the highest leaf number assigned to the oldest leaf(at the bottom).

A population of tobacco plants or a collection of tobacco leaves fordetermining an average measurement (e.g., alkaloid or nicotine level orleaf grading) can be of any size, for example, 5, 10, 15, 20, 25, 30,35, 40, or 50. Industry-accepted standard protocols are followed fordetermining average measurements or grad index values.

As used herein, “topping” refers to the removal of the stalk apex,including the SAM, flowers, and up to several adjacent leaves, when atobacco plant is near vegetative maturity and around the start ofreproductive growth. Typically, tobacco plants are topped in the buttonstage (soon after the flower begins to appear). For example, greenhouseor field-grown tobacco plants can be topped when 50% of the plants haveat least one open flower. Topping a tobacco plant results in the loss ofapical dominance and also induces increased alkaloid production.

Unless indicated otherwise, the nicotine or alkaloid level (or anotherleaf chemistry or property characterization) of a tobacco plant ismeasured 2 weeks after topping. Alternatively, other time points can beused. In an aspect, the nicotine or alkaloid level (or another leafchemistry or property characterization) of a tobacco plant is measuredabout 1, 2, 3, 4, or 5 weeks after topping. In another aspect, thenicotine, alkaloid, or polyamine level (or another leaf chemistry orproperty characterization) of a tobacco plant is measured about 3, 5, 7,10, 12, 14, 17, 19 or 21 days after topping.

As used herein, “similar growth conditions” or “comparable growthconditions” refer to similar environmental conditions and/or agronomicpractices for growing and making meaningful comparisons between two ormore plant genotypes so that neither environmental conditions noragronomic practices would contribute to or explain any differenceobserved between the two or more plant genotypes. Environmentalconditions include, for example, light, temperature, water (humidity),and nutrition (e.g., nitrogen and phosphorus). Agronomic practicesinclude, for example, seeding, clipping, undercutting, transplanting,topping, and suckering. See Chapters 4B and 4C of Tobacco, Production,Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing,Oxford (1999), pp 70-103.

“Alkaloids” are complex, nitrogen-containing compounds that naturallyoccur in plants, and have pharmacological effects in humans and animals.“Nicotine” is the primary natural alkaloid in commercialized cigarettetobacco and accounts for about 90 percent of the alkaloid content inNicotiana tabacum. Other major alkaloids in tobacco include cotinine,nornicotine, myosmine, nicotyrine, anabasine and anatabine. Minortobacco alkaloids include nicotine-n-oxide, N-methyl anatabine, N-methylanabasine, pseudooxynicotine, 2,3 dipyridyl and others.

Alkaloid levels can be assayed by methods known in the art, for exampleby quantification based on gas-liquid chromatography, high performanceliquid chromatography, radio-immunoassays, and enzyme-linkedimmunosorbent assays. For example, nicotinic alkaloid levels can bemeasured by a GC-FID method based on CORESTA Recommended Method No. 7,1987 and ISO Standards (ISO TC 126N 394 E. See also Hibi et al., PlantPhysiology 100: 826-35 (1992) for a method using gas-liquidchromatography equipped with a capillary column and an FID detector.

Unless specifically indicated otherwise, alkaloids and nicotine levelsare measured using a method in accordance with CORESTA Method No 62,Determination of Nicotine in Tobacco and Tobacco Products by GasChromatographic Analysis, February 2005, and those defined in theCenters for Disease Control and Prevention's Protocol for Analysis ofNicotine, Total Moisture and pH in Smokeless Tobacco Products, aspublished in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and asamended in Vol. 74, No. 4, Jan. 7, 2009). Alternatively, tobacco totalalkaloids can be measured using a segmented-flow colorimetric methoddeveloped for analysis of tobacco samples as adapted by SkalarInstrument Co (West Chester, Pa.) and described by Collins et al.,Tobacco Science 13:79-81 (1969). In short, samples of tobacco can bedried, ground, and extracted prior to analysis of total alkaloids andreducing sugars. The method then employs an acetic acid/methanol/waterextraction and charcoal for decolorization. Determination of totalalkaloids is based on the reaction of cyanogen chloride with nicotinealkaloids in the presence of an aromatic amine to form a colored complexwhich is measured at 460 nm. Unless specified otherwise, total alkaloidlevels or nicotine levels shown herein are on a dry weight basis (e.g.,percent total alkaloid or percent nicotine).

In an aspect, a tobacco plant comprises an average nicotine or totalalkaloid level selected from the group consisting of about 0.01%, 0.02%,0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%,2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%,3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and9% on a dry weight basis. In another aspect, a tobacco plant comprisesan average nicotine or total alkaloid level selected from the groupconsisting of about between 0.01% and 0.02%, between 0.02% and 0.05%,between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%,between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%,between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%,between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%,between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1%and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%,between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%,between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis.In a further aspect, a tobacco plant comprises an average nicotine ortotal alkaloid level selected from the group consisting of about between0.01% and 0.1%, between 0.02% and 0.2%, between 0.03% and 0.3%, between0.04% and 0.4%, between 0.05% and 0.5%, between 0.75% and 1%, between0.1% and 1.5%, between 0.15% and 2%, between 0.2% and 3%, and between0.3% and 3.5% on a dry weight basis.

The present disclosure also provides a tobacco plant having an alterednicotine level without negative impacts over other tobacco traits, e.g.,leaf grade index value. In an aspect, a low-nicotine or nicotine-freetobacco variety provides cured tobacco of commercially acceptable grade.Tobacco grades are evaluated based on factors including, but not limitedto, the leaf stalk position, leaf size, leaf color, leaf uniformity andintegrity, ripeness, texture, elasticity, sheen (related with theintensity and the depth of coloration of the leaf as well as the shine),hygroscopicity (the faculty of the tobacco leaves to absorb and toretain the ambient moisture), and green nuance or cast. Leaf grade canbe determined, for example, using an Official Standard Grade publishedby the Agricultural Marketing Service of the US Department ofAgriculture (7 U.S.C. § 511). See, e.g., Official Standard Grades forBurley Tobacco (U.S. Type 31 and Foreign Type 93), effective Nov. 5,1990 (55 F.R. 40645); Official Standard Grades for Flue-Cured Tobacco(U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective Mar. 27, 1989(54 F.R. 7925); Official Standard Grades for Pennsylvania SeedleafTobacco (U.S. Type 41), effective Jan. 8, 1965 (29 F.R. 16854); OfficialStandard Grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44),effective Dec. 8, 1963 (28 F.R. 11719 and 28 F.R. 11926); OfficialStandard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Gradesfor Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effectiveNov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Georgia andFlorida Shade-Grown Cigar-Wrapper Tobacco (U. S. Type 62), EffectiveApril 1971. A USDA grade index value can be determined according to anindustry accepted grade index. See, e.g., Bowman et al, Tobacco Science,32:39-40(1988); Legacy Tobacco Document Library (Bates Document#523267826-523267833, Jul. 1, 1988, Memorandum on the Proposed BurleyTobacco Grade Index); and Miller et al., 1990, Tobacco Intern.,192:55-57 (all foregoing references are incorporated by inference intheir entirety). In an aspect, a USDA grade index is a 0-100 numericalrepresentation of federal grade received and is a weighted average ofall stalk positions. A higher grade index indicates higher quality.Alternatively, leaf grade can be determined via hyper-spectral imaging.See e.g., WO 2011/027315 (published on Mar. 10, 2011, and incorporatedby inference in its entirety).

In an aspect, a tobacco plant provided herein comprises a similar levelof one or more tobacco aroma compounds compared to a control tobaccoplant when grown in similar growth conditions. In another aspect, atobacco plant provided herein comprise a similar level of one or moretobacco aroma compounds selected from the group consisting of3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, acembrenoid, a sugar ester, and a reducing sugar, compared to a controltobacco plant when grown in similar growth conditions.

As used herein, tobacco aroma compounds are compounds associated withthe flavor and aroma of tobacco smoke. These compounds include, but arenot limited to, 3-methylvaleric acid, valeric acid, isovaleric acid,cembrenoid and labdenoid diterpenes, and sugar esters. Concentrations oftobacco aroma compounds can be measured by any known metaboliteprofiling methods in the art including, without limitation, gaschromatography mass spectrometry (GC-MS), Nuclear Magnetic ResonanceSpectroscopy, liquid chromatography-linked mass spectrometry. See TheHandbook of Plant Metabolomics, edited by Weckwerth and Kahl,(Wiley-Blackwell) (May 28, 2013).

As used herein, “reducing sugar(s)” are any sugar (monosaccharide orpolysaccharide) that has a free or potentially free aldehyde or ketonegroup. Glucose and fructose act as nicotine buffers in cigarette smokeby reducing smoke pH and effectively reducing the amount of “free”unprotonated nicotine. Reducing sugars balances smoke flavor, forexample, by modifying the sensory impact of nicotine and other tobaccoalkaloids. An inverse relationship between sugar content and alkaloidcontent has been reported across tobacco varieties, within the samevariety, and within the same plant line caused by planting conditions.Reducing sugar levels can be measured using a segmented-flowcolorimetric method developed for analysis of tobacco samples as adaptedby Skalar Instrument Co (West Chester, Pa.) and described by Davis,Tobacco Science 20:139-144 (1976). For example, a sample is dialyzedagainst a sodium carbonate solution. Copper neocuproin is added to thesample and the solution is heated. The copper neocuproin chelate isreduced in the presence of sugars resulting in a colored complex whichis measured at 460 nm.

In an aspect, a tobacco plant comprises one or more non-naturallyexisting mutant alleles in one or more PMT gene loci which reduce oreliminate PMT enzymatic activity from the one or more PMT gene loci. Inan aspect, these mutant alleles result in lower nicotine levels. Mutantpmt alleles can be introduced by any method known in the art includingrandom or targeted mutagenesis approaches.

Such mutagenesis methods include, without limitation, treatment of seedswith ethyl methylsulfate (EMS) (Hildering and Verkerk, In, The use ofinduced mutations in plant breeding. Pergamon press, pp 317-320, 1965)or UV-irradiation, X-rays, and fast neutron irradiation (see, forexample, Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; and Poehlman,Breeding Field Crops, Van Nostrand Reinhold, N.Y. (3.sup.rd ed), 1987),transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and5,013,658), as well as T-DNA insertion methodologies (Hoekema et al.,1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists ofchemically inducing random point mutations over the length of thegenome. Fast neutron mutagenesis consists of exposing seeds to neutronbombardment which causes large deletions through double stranded DNAbreakage. Transposon tagging comprises inserting a transposon within anendogenous gene to reduce or eliminate expression of the gene. The typesof mutations that may be present in a tobacco gene include, for example,point mutations, deletions, insertions, duplications, and inversions.Such mutations desirably are present in the coding region of a tobaccogene; however mutations in the promoter region, and intron, or anuntranslated region of a tobacco gene may also be desirable.

In addition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the present disclosure. See, McCallumet al. (2000) Nat. Biotechnol. 18:455-457. Mutations that impact geneexpression or that interfere with the function of genes can bedetermined using methods that are well known in the art. Insertionalmutations in gene exons usually result in null-mutants. Mutations inconserved residues can be particularly effective in inhibiting thefunction of a protein. In an aspect, tobacco plants comprise a nonsense(e.g., stop codon) mutation in one or more PMT genes described herein.

It will be appreciated that, when identifying a mutation, the endogenousreference DNA sequence should be from the same variety of tobacco. Forexample, if a modified tobacco plant comprising a mutation is from thevariety TN90, then the endogenous reference sequence must be theendogenous TN90 sequence, not a homologous sequence from a differenttobacco variety (e.g., K326). Similarly, if a modified tobacco cellcomprising a mutation is a TN90 cell, then the endogenous referencesequence must be the endogenous TN90 sequence, not a homologous sequencefrom a tobacco cell from a different tobacco variety (e.g., K326).

In an aspect, the present disclosure also provides a tobacco line withaltered nicotine levels while maintaining commercially acceptable leafquality. This line can be produced by introducing mutations into one ormore PMT genes via precise genome engineering technologies, for example,Transcription activator-like effector nucleases (TALENs), meganuclease,zinc finger nuclease, and a clustered regularly-interspaced shortpalindromic repeats (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, aCRISPR/Csm1 system, and a combination thereof (see, for example, U.S.Patent Application publication 2017/0233756). See, e.g., Gaj et al.,Trends in Biotechnology, 31(7):397-405 (2013).

The screening and selection of mutagenized tobacco plants can be throughany methodologies known to those having ordinary skill in the art.Examples of screening and selection methodologies include, but are notlimited to, Southern analysis, PCR amplification for detection of apolynucleotide, Northern blots, RNase protection, primer-extension,RT-PCR amplification for detecting RNA transcripts, Sanger sequencing,Next Generation sequencing technologies (e.g., Illumina, PacBio, IonTorrent, 454), enzymatic assays for detecting enzyme or ribozymeactivity of polypeptides and polynucleotides, and protein gelelectrophoresis, Western blots, immunoprecipitation, and enzyme-linkedimmunoassays to detect polypeptides. Other techniques such as in situhybridization, enzyme staining, and immunostaining also can be used todetect the presence or expression of polypeptides and/orpolynucleotides. Methods for performing all of the referenced techniquesare known.

In an aspect, a tobacco plant or plant genome provided herein is mutatedor edited by a nuclease selected from the group consisting of ameganuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a CRISPR/Cas9 nuclease, aCRISPR/Cpf1 nuclease, or a CRISPR/Csm1 nuclease.

As used herein, “editing” or “genome editing” refers to targetedmutagenesis of at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, or at least 10nucleotides of an endogenous plant genome nucleic acid sequence, orremoval or replacement of an endogenous plant genome nucleic acidsequence. In an aspect, an edited nucleic acid sequence provided has atleast 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%,at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, atleast 91%, at least 90%, at least 85%, at least 80%, or at least 75%sequence identity with an endogenous nucleic acid sequence. In anaspect, an edited nucleic acid sequence provided has at least 99.9%, atleast 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, atleast 95%, at least 94%, at least 93%, at least 92%, at least 91%, atleast 90%, at least 85%, at least 80%, or at least 75% sequence identitywith a polynucleotide selected from the group consisting of SEQ ID NOs:1 to 10, and fragments thereof. In another aspect, an edited nucleicacid sequence provided has at least 99.9%, at least 99.5%, at least 99%,at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, atleast 93%, at least 92%, at least 91%, at least 90%, at least 85%, atleast 80%, or at least 75% sequence identity with a polynucleotideencoding a polypeptide selected from the group consisting of SEQ ID NOs:11 to 15.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1 and CRISPR/Cpf1induce a double-strand DNA break at a target site of a genomic sequencethat is then repaired by the natural processes of homologousrecombination (HR) or non-homologous end-joining (NHEJ). Sequencemodifications then occur at the cleaved sites, which can includedeletions or insertions that result in gene disruption in the case ofNHEJ, or integration of donor nucleic acid sequences by HR. In anaspect, a method provided comprises editing a plant genome with anuclease provided to mutate at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, or more than 10 nucleotides in the plant genome via HR with a donorpolynucleotide. In an aspect, a mutation provided is caused by genomeediting using a nuclease. In another aspect, a mutation provided iscaused by non-homologous end-joining or homologous recombination.

Meganucleases, which are commonly identified in microbes, are uniqueenzymes with high activity and long recognition sequences (>14 bp)resulting in site-specific digestion of target DNA. Engineered versionsof naturally occurring meganucleases typically have extended DNArecognition sequences (for example, 14 to 40 bp). The engineering ofmeganucleases can be more challenging than that of ZFNs and TALENsbecause the DNA recognition and cleavage functions of meganucleases areintertwined in a single domain. Specialized methods of mutagenesis andhigh-throughput screening have been used to create novel meganucleasevariants that recognize unique sequences and possess improved nucleaseactivity.

ZFNs are synthetic proteins consisting of an engineered zinc fingerDNA-binding domain fused to the cleavage domain of the FokI restrictionendonuclease. ZFNs can be designed to cleave almost any long stretch ofdouble-stranded DNA for modification of the zinc finger DNA-bindingdomain. ZFNs form dimers from monomers composed of a non-specific DNAcleavage domain of FokI endonuclease fused to a zinc finger arrayengineered to bind a target DNA sequence.

The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-fingerarrays. The amino acids at positions −1, +2, +3, and +6 relative to thestart of the zinc finger ∞-helix, which contribute to site-specificbinding to the target DNA, can be changed and customized to fit specifictarget sequences. The other amino acids form the consensus backbone togenerate ZFNs with different sequence specificities. Rules for selectingtarget sequences for ZFNs are known in the art.

The FokI nuclease domain requires dimerization to cleave DNA andtherefore two ZFNs with their C-terminal regions are needed to bindopposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFNmonomer can cute the target site if the two-ZF-binding sites arepalindromic. The term ZFN, as used herein, is broad and includes amonomeric ZFN that can cleave double stranded DNA without assistancefrom another ZFN. The term ZFN is also used to refer to one or bothmembers of a pair of ZFNs that are engineered to work together to cleaveDNA at the same site.

Without being limited by any scientific theory, because the DNA-bindingspecificities of zinc finger domains can in principle be re-engineeredusing one of various methods, customized ZFNs can theoretically beconstructed to target nearly any gene sequence. Publicly availablemethods for engineering zinc finger domains include Context-dependentAssembly (CoDA), Oligomerized Pool Engineering (OPEN), and ModularAssembly.

TALENs are artificial restriction enzymes generated by fusing thetranscription activator-like effector (TALE) DNA binding domain to aFokI nuclease domain. When each member of a TALEN pair binds to the DNAsites flanking a target site, the FokI monomers dimerize and cause adouble-stranded DNA break at the target site. The term TALEN, as usedherein, is broad and includes a monomeric TALEN that can cleave doublestranded DNA without assistance from another TALEN. The term TALEN isalso used to refer to one or both members of a pair of TALENs that worktogether to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bindpractically any DNA sequence. TALE proteins are DNA-binding domainsderived from various plant bacterial pathogens of the genus Xanthomonas.The Xanthomonas pathogens secrete TALEs into the host plant cell duringinfection. The TALE moves to the nucleus, where it recognizes and bindsto a specific DNA sequence in the promoter region of a specific DNAsequence in the promoter region of a specific gene in the host genome.TALE has a central DNA-binding domain composed of 13-28 repeat monomersof 33-34 amino acids. The amino acids of each monomer are highlyconserved, except for hypervariable amino acid residues at positions 12and 13. The two variable amino acids are called repeat-variablediresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDspreferentially recognize adenine, thymine, cytosine, andguanine/adenine, respectively, and modulation of RVDs can recognizeconsecutive DNA bases. This simple relationship between amino acidsequence and DNA recognition has allowed for the engineering of specificDNA binding domains by selecting a combination of repeat segmentscontaining the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokIcleavage domain with mutations have been designed to improve cleavagespecificity and cleavage activity. The FokI domain functions as a dimer,requiring two constructs with unique DNA binding domains for sites inthe target genome with proper orientation and spacing. Both the numberof amino acid residues between the TALEN DNA binding domain and the FokIcleavage domain and the number of bases between the two individual TALENbinding sites are parameters for achieving high levels of activity.

A relationship between amino acid sequence and DNA recognition of theTALE binding domain allows for designable proteins. Software programssuch as DNA Works can be used to design TALE constructs. Other methodsof designing TALE constructs are known to those of skill in the art. SeeDoyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak etal., Nucleic Acids Research (2011). 39:e82; andtale-nt.cac.cornell.edu/about.

A CRISPR/Cas9 system, CRISPR/Csm1, or a CRISPR/Cpf1 system arealternatives to the FokI-based methods ZFN and TALEN. The CRISPR systemsare based on RNA-guided engineered nucleases that use complementary basepairing to recognize DNA sequences at target sites.

CRISPR/Cas9, CRISPR/Csm1, and a CRISPR/Cpf1 systems are part of theadaptive immune system of bacteria and archaea, protecting them againstinvading nucleic acids such as viruses by cleaving the foreign DNA in asequence-dependent manner. The immunity is acquired by the integrationof short fragments of the invading DNA known as spacers between twoadjacent repeats at the proximal end of a CRISPR locus. The CRISPRarrays, including the spacers, are transcribed during subsequentencounters with invasive DNA and are processed into small interferingCRISPR RNAs (crRNAs) approximately 40 nt in length, which combine withthe trans-activating CRISPR RNA (tracrRNA) to activate and guide theCas9 nuclease. This cleaves homologous double-stranded DNA sequencesknown as protospacers in the invading DNA. A prerequisite for cleavageis the presence of a conserved protospacer-adjacent motif (PAM)downstream of the target DNA, which usually has the sequence 5-NGG-3 butless frequently NAG. Specificity is provided by the so-called “seedsequence” approximately 12 bases upstream of the PAM, which must matchbetween the RNA and target DNA. Cpf1 and Csm1 act in a similar manner toCas9, but Cpf1 and Csm1 do not require a tracrRNA.

In still another aspect, a tobacco plant provided here comprises one ormore pint mutations and further comprises one or more mutations in oneor more loci encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5,CYP82E10) that confer reduced amounts of nornicotine (See U.S. Pat. Nos.8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706)compared to a control plant lacking one or more mutations in one or moreloci encoding a nicotine demethylase. In an aspect, a tobacco plantdescribed further comprises reduced nicotine demethylase activitycompared to a control plant when grown and cured under comparableconditions.

In an aspect, a pint mutant tobacco plant further comprises a mutationcapable of producing a leaf comprising an anabasine level less than theanabasine level of a leaf from a wild-type control tobacco plant whengrown and processed under comparable conditions. In another aspect, apint mutant tobacco plant further comprises a mutation capable ofproducing a leaf comprising an anabasine level less than 5%, 10%, 20%,25%, 30%, 40%, 50%, 60%, 70%, or 80% of the anabasine level of a leaffrom a wild-type control tobacco plant when grown and processed undercomparable conditions.

In an aspect, a pint mutant tobacco plant comprises a further mutationcapable of producing a leaf comprising a more than 2 fold reduction ofthe anatabine level compared to a leaf from a control tobacco plant whengrown and processed under comparable conditions. In another aspect, apint mutant tobacco plant comprises a further mutation capable ofproducing a leaf comprising a more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13 fold reduction of the anatabine level compared to a leaf from awild-type control tobacco plant when grown and processed undercomparable conditions. In an aspect, a mutation providing lower level ofanatabine is a mutation described in US Application Publication No.2014/0283165 and US Application Publication No. 2016/0010103. In anotheraspect, a pint mutant further comprises a mutation in a quinolatephosphoribosyl transferase (QPT) or quinolinate synthase (QS) gene. In afurther aspect, a pmt mutant plant further comprises a transgene ormutation suppressing the expression or activity of a QPT or QS gene.

In an aspect, a pint mutant tobacco plant further comprises a mutationcapable of providing a nornicotine level less than 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, or 35% of the nornicotine level of a leaf froma wild-type control tobacco plant when grown and processed undercomparable conditions.

In an aspect, a pmt mutant tobacco plant is capable of producing a curedleaf comprising a total N-nitrosonornicotine (NNN) level of less than 2,less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, lessthan 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6,less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than0.15, less than 0.1, or less than 0.05 ppm.

In another aspect, a pmt mutant tobacco plant is capable of producing acured leaf comprising a total NNN level of between 2 and 0.05, between1.9 and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6and 0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and0.05, between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05,between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05,between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05,between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, orbetween 0.1 and 0.05 parts per million (ppm).

In an aspect, a pmt mutant tobacco plant is capable of producing a curedleaf comprising a total nicotine-derived nitrosamine ketone (NNK) levelof less than 2, less than 1.9, less than 1.8, less than 1.7, less than1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, lessthan 1.1, less than 1.0, less than 0.9, less than 0.8, less than 0.7,less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than0.2, less than 0.15, less than 0.1, or less than 0.05 ppm.

In another aspect, a pmt mutant tobacco plant is capable of producing acured leaf comprising a total NNK level of between 2 and 0.05, between1.9 and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6and 0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and0.05, between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05,between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05,between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05,between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, orbetween 0.1 and 0.05 ppm.

In an aspect, a pint mutant tobacco plant further comprises a mutationor transgene providing an increased level of one or more antioxidants.In another aspect, a pmt mutant tobacco plant further comprises agenetic modification in an endogenous gene and further comprises anincreased level of one or more antioxidants in a cured leaf compared toa control cured tobacco leaf lacking the genetic modification, where theendogenous gene encodes an antioxidant biosynthetic enzyme, a regulatorytranscription factor of an antioxidant, an antioxidant transporter, anantioxidant metabolic enzyme, or a combination thereof. In a furtheraspect, a pint mutant tobacco plant further comprises a transgene andfurther comprises an increased level of one or more antioxidants in acured leaf compared to a control cured tobacco leaf lacking thetransgene, where the transgene encodes or directly modulates anantioxidant biosynthetic enzyme, a regulatory transcription factor of anantioxidant, an antioxidant transporter, an antioxidant metabolicenzyme, or a combination thereof. In an aspect, a pint mutant tobaccoplant further comprises a transgene or a cisgenic construct expressingone or more genes selected from the group consisting of AtPAP1, NtAN2,NtAN1, NtJAF13, NtMyb3, chorismate mutase, and arogenate dehydrotase(ADT). In another aspect, a pint mutant tobacco plant further comprisesone or more transgenes or genetic modification for increasingantioxidants or decreasing one or more TSNAs as described in WIPOPublication No. 2018/067985 or US Publication No. 2018/0119163.

In an aspect, a tobacco plant described is a modified tobacco plant. Asused herein, “modified”, in the context of a plant, refers to a plantcomprising a genetic alteration introduced for certain purposes andbeyond natural polymorphisms.

In an aspect, a tobacco plant described is a cisgenic plant. As usedherein, “cisgenesis” or “cisgenic” refers to genetic modification of aplant, plant cell, or plant genome in which all components (e.g.,promoter, donor nucleic acid, selection gene) have only plant origins(i.e., no non-plant origin components are used). In an aspect, a plant,plant cell, or plant genome provided is cisgenic. Cisgenic plants, plantcells, and plant genomes provided can lead to ready-to-use tobaccolines. In another aspect, a tobacco plant provided comprises nonon-tobacco genetic material or sequences.

As used herein, “gene expression” or expression of a gene refers to thebiosynthesis or production of a gene product, including thetranscription and/or translation of the gene product.

In an aspect, a tobacco plant provided comprises one or more pmtmutations and further comprises reduced expression or activity of one ormore genes involved in nicotine biosynthesis or transport. Genesinvolved in nicotine biosynthesis include, but are not limited to,arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADHdehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilateisomerase (PRAI), quinolate phosphoribosyl transferase (QPT), andS-adenosyl-methionine synthetase (SAMS). Nicotine Synthase, whichcatalyzes the condensation step between a nicotinic acid derivative andmethylpyrrolinium cation, has not been elucidated although two candidategenes (A622 and NBB1) have been proposed. See US 2007/0240728 A1 and US2008/0120737A1. A622 encodes an isoflavone reductase-like protein. Inaddition, several transporters may be involved in the translocation ofnicotine. A transporter gene, named MATE, has been cloned andcharacterized (Morita et al., PNAS 106:2447-52 (2009)).

In an aspect, a tobacco plant provided comprises one or more pmtmutations and further comprises a reduced level of mRNA, protein, orboth of one or more genes encoding a product selected from the groupconsisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1,compared to a control tobacco plant. In another aspect, a tobacco plantsprovided comprises one or more pint mutations and further comprises atransgene directly suppressing the expression of one or more genesencoding a product selected from the group consisting of MPO, QPT, ADC,ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, a tobaccoplant provided comprises one or more pmt mutations and further comprisesa transgene or mutation suppressing the expression or activity of one ormore genes encoding a product selected from the group consisting of MPO,QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

In an aspect, a tobacco plant provided is from a tobacco type selectedfrom the group consisting of flue-cured tobacco, air-cured tobacco, darkair-cured tobacco, dark fire-cured tobacco, Galpao tobacco, and Orientaltobacco. In another aspect, a tobacco plant provided is from a tobaccotype selected from the group consisting of Burley tobacco, Marylandtobacco, and dark tobacco.

In an aspect, a tobacco plant provided is in a flue-cured tobaccobackground or exhibits one or more flue-cured tobacco characteristicdescribed here. Flue-cured tobaccos (also called Virginia or brighttobaccos) amount to approximately 40% of world tobacco production.Flue-cured tobaccos are often also referred to as “bright tobacco”because of the golden-yellow to deep-orange color it reaches duringcuring. Flue-cured tobaccos have a light, bright aroma and taste.Flue-cured tobaccos are generally high in sugar and low in oils. Majorflue-cured tobacco growing countries are Argentina, Brazil, China,India, Tanzania and the U.S. In an aspect, a low-alkaloid orlow-nicotine tobacco plant or seed provided is in a flue-cured tobaccobackground selected from the group consisting of CC 13, CC 27, CC 33, CC37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399,K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety essentiallyderived from any one of the foregoing varieties. In another aspect, alow-alkaloid or low-nicotine tobacco plant or seed provided is in aflue-cured tobacco background selected from the group consisting ofCoker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF,NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713,Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51,Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108,Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va116, Va 182, and any variety essentially derived from any one of theforegoing varieties. See WO 2004/041006 A1. In a further aspect,low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties,or lines are in any flue cured background selected from the groupconsisting of K326, K346, and NC 196.

In an aspect, a tobacco plant provided is in an air-cured tobaccobackground or exhibits one or more air-cured tobacco characteristicdescribed here. Air-cured tobaccos include Burley, Md., and darktobaccos. The common factor is that curing is primarily withoutartificial sources of heat and humidity. Burley tobaccos are light todark brown in color, high in oil, and low in sugar. Burley tobaccos areair-cured in barns. Major Burley growing countries are Argentina,Brazil, Italy, Malawi, and the U.S. Maryland tobaccos are extremelyfluffy, have good burning properties, low nicotine and a neutral aroma.Major Maryland growing countries include the U.S. and Italy. In anaspect, a low-alkaloid or low-nicotine tobacco plant or seed provided isin a Burley tobacco background selected from the group consisting ofClay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712,NCBH 129, Bu 21×Ky 10, HBO4P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561,Pf561 and Va 509. In a further aspect, low-alkaloid or low-nicotinetobacco plants, seeds, hybrids, varieties, or lines are in any Burleybackground selected from the group consisting of TN 90, KT 209, KT 206,KT212, and HB 4488. In another aspect, a low-alkaloid or low-nicotinetobacco plant or seed provided is in a Maryland tobacco backgroundselected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md872 and Md 341.

In an aspect, a tobacco plant provided is in a dark air-cured tobaccobackground or exhibits one or more dark air-cured tobacco characteristicdescribed here. Dark air-cured tobaccos are distinguished from othertypes primarily by its curing process which gives dark air-cured tobaccoits medium- to dark-brown color and distinct aroma. Dark air-curedtobaccos are mainly used in the production of chewing tobacco and snuff.In an aspect, a low-alkaloid or low-nicotine tobacco plant or seedprovided is in a dark air-cured tobacco background selected from thegroup consisting of Sumatra, Jatim, Dominican Cubano, Besuki, Onesucker, Green River, Va. sun-cured, and Paraguan Passado.

In an aspect, a tobacco plant provided is in a dark fire-cured tobaccobackground or exhibits one or more dark fire-cured tobaccocharacteristic described here. Dark fire-cured tobaccos are generallycured with low-burning wood fires on the floors of closed curing barns.Their leaves have low sugar content but high nicotine content. Darkfire-cured tobaccos are used for making pipe blends, cigarettes, chewingtobacco, snuff and strong-tasting cigars. Major growing regions for darkfire-cured tobaccos are Tennessee, Kentucky, and Virginia, USA. In anaspect, a low-alkaloid or low-nicotine tobacco plant or seed provided isin a dark fire-cured tobacco background selected from the groupconsisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole,Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, SmallStalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TND94, TN D950, VA 309, and VA 359.

In an aspect, a tobacco plant provided is in an Oriental tobaccobackground or exhibits one or more Oriental tobacco characteristicdescribed here. Oriental tobaccos are also referred to as Greek, aromaand Turkish tobaccos due to the fact that they are typically grown ineastern Mediterranean regions such as Turkey, Greece, Bulgaria,Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leafsize, characteristic of today's Oriental varieties, as well as itsunique aroma properties are a result of the plant's adaptation to thepoor soil and stressful climatic conditions in which it develop overmany past centuries. In an aspect, a low-alkaloid or low-nicotinetobacco plant or seed provided is in an Oriental tobacco backgroundselected from the group consisting of Izmir, Katerini, Samsun, Basma andKrumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne,Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra,Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any varietyessentially derived from any one of the foregoing varieties.

In an aspect, low-alkaloid or low-nicotine tobacco plants, seeds,hybrids, varieties, or lines are essentially derived from or in thegenetic background of BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600,GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399,K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944,msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297,NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (TomRosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC,KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC,KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC,PD7312LC, ShireyLC, or any commercial tobacco variety according tostandard tobacco breeding techniques known in the art.

All foregoing mentioned specific varieties of dark air-cured, Burley,Md., dark fire-cured, or Oriental type are listed only for exemplarypurposes. Any additional dark air-cured, Burley, Md., dark fire-cured,Oriental varieties are also contemplated in the present application.

Also provided are populations of tobacco plants described. In an aspect,a population of tobacco plants has a planting density of between about5,000 and about 8000, between about 5,000 and about 7,600, between about5,000 and about 7,200, between about 5,000 and about 6,800, betweenabout 5,000 and about 6,400, between about 5,000 and about 6,000,between about 5,000 and about 5,600, between about 5,000 and about5,200, between about 5,200 and about 8,000, between about 5,600 andabout 8,000, between about 6,000 and about 8,000, between about 6,400and about 8,000, between about 6,800 and about 8,000, between about7,200 and about 8,000, or between about 7,600 and about 8,000 plants peracre. In another aspect, a population of tobacco plants is in a soiltype with low to medium fertility.

Also provided are containers of seeds from tobacco plants described. Acontainer of tobacco seeds of the present disclosure may contain anynumber, weight, or volume of seeds. For example, a container can containat least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds.Alternatively, the container can contain at least, or greater than,about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4pounds, 5 pounds or more seeds. Containers of tobacco seeds may be anycontainer available in the art. By way of non-limiting example, acontainer may be a box, a bag, a packet, a pouch, a tape roll, a tube,or a bottle.

Also provided is cured tobacco material made from a low-alkaloid orlow-nicotine tobacco plant described. Further provided is cured tobaccomaterial made from a tobacco plant described with higher levels of totalalkaloid or nicotine.

“Curing” is the aging process that reduces moisture and brings about thedestruction of chlorophyll giving tobacco leaves a golden color and bywhich starch is converted to sugar. Cured tobacco therefore has a higherreducing sugar content and a lower starch content compared to harvestedgreen leaf. In an aspect, green leaf tobacco provided can be cured usingconventional means, e.g., flue-cured, barn-cured, fire-cured, air-curedor sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford) for a description of different types of curingmethods. Cured tobacco is usually aged in a wooden drum (e.g., ahogshead) or cardboard cartons in compressed conditions for severalyears (e.g., two to five years), at a moisture content ranging from 10%to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured andaged tobacco then can be further processed. Further processing includesconditioning the tobacco under vacuum with or without the introductionof steam at various temperatures, pasteurization, and fermentation.Fermentation typically is characterized by high initial moisturecontent, heat generation, and a 10 to 20% loss of dry weight. See, e.g.,U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S.Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cure, aged, and fermented tobacco can be furtherprocessed (e.g., cut, shredded, expanded, or blended). See, for example,U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In an aspect, thecured tobacco material of the present disclosure is sun-cured. Inanother aspect, the cured tobacco material of the present disclosure isflue-cured, air-cured, or fire-cured.

The presence of mold on cured tobacco can significantly reduce thequality and marketability (e.g., leaf grade) of the cured leaves. Moldgrowth is a common problem that can occur during extended periods ofhigh humidity (e.g., greater than 70% relative humidity) at temperaturesbetween approximately 10° C. (50° F.) and 32.2° C. (90° F.). Mold tendsto be more prevalent at higher temperatures.

Tobacco plants, varieties, and lines provided herein comprising a mutantallele in one or more PMT genes, two or more PMT genes, three or morePMT genes, four or more PMT genes, or five PMT genes exhibit reducedmold infection as compared to the low alkaloid tobacco variety LA Burley21 (LA BU 21). Similarly, tobacco plants, varieties, and lines providedherein comprising an RNAi construct that downregulates expression ortranslation of one or more PMT genes, two or more PMT genes, three ormore PMT genes, four or more PMT genes, or five PMT genes exhibitreduced mold infection as compared to the low alkaloid tobacco varietyLA Burley 21 (LA BU 21).

LA BU 21 is a low total alkaloid tobacco line produced by incorporationof a low alkaloid gene(s) from a Cuban cigar variety into Burley 21through several backcrosses (Legg et al., Crop Science, 10:212 (1970)).It has approximately 0.2% total alkaloids (dry weight) compared to theabout 3.5% (dry weight) of its parent, Burley 21. LA B U 21 has a leafgrade well below commercially acceptable standards.

In an aspect, a cured tobacco leaf comprising a mutant allele of pmt1acomprises no observable mold infection. In another aspect, a curedtobacco leaf comprising a mutant allele of pmt1b comprises no observablemold infection. In another aspect, a cured tobacco leaf comprising amutant allele of pmt2 comprises no observable mold infection. In anotheraspect, a cured tobacco leaf comprising a mutant allele of pmt3comprises no observable mold infection. In another aspect, a curedtobacco leaf comprising a mutant allele of pmt4 comprises no observablemold infection. In another aspect, a cured tobacco leaf comprising amutant allele of pmt1a, a mutant allele of pmt1b, a mutant allele ofpmt2, a mutant allele of pmt3, and a mutant allele of pmt4 comprises noobservable mold infection.

In an aspect, a cured tobacco leaf comprising a mutant allele of pmt1acomprises a reduced mold infection as compared to a control curedtobacco leaf from the variety LA BU 21. In another aspect, a curedtobacco leaf comprising a mutant allele of pmt1b comprises a reducedmold infection as compared to a control cured tobacco leaf from thevariety LA BU 21. In another aspect, a cured tobacco leaf comprising amutant allele of pmt2 comprises a reduced mold infection as compared toa control cured tobacco leaf from the variety LA BU 21. In anotheraspect, a cured tobacco leaf comprising a mutant allele of pmt3comprises a reduced mold infection as compared to a control curedtobacco leaf from the variety LA BU 21. In another aspect, a curedtobacco leaf comprising a mutant allele of pmt4 comprises a reduced moldinfection as compared to a control cured tobacco leaf from the varietyLA BU 21. In another aspect, a cured tobacco leaf comprising a mutantallele of pmt1a, a mutant allele of pmt1b, a mutant allele of pmt2, amutant allele of pmt3, and a mutant allele of pmt4 comprises a reducedmold infection as compared to a control cured tobacco leaf from thevariety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or lineprovided in any one of Tables 4A to 4E, Table 10, or Table 14 comprisesno observable mold infection. In another aspect, a cured leaf from atobacco plant, variety, or line provided in any one of Tables 4A to 4E,Table 10, or Table 14 comprises a reduced mold infection as compared toa control cured tobacco leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or linecomprising one or more pint mutations provided in any one of Tables 5Ato 5E and Tables 12A to 12E comprises no observable mold infection. Inanother aspect, a cured leaf from a tobacco plant, variety, or linecomprising one or more pmt mutations provided in any one of Tables 5A to5E and Tables 12A to 12E comprises a reduced mold infection as comparedto a control cured leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or linecomprising a mutant allele of pmt1a comprises a higher leaf grade than acontrol cured leaf from the variety LA BU 21. In an aspect, a cured leaffrom a tobacco plant, variety, or line comprising a mutant allele ofpmt1b comprises a higher leaf grade than a control cured leaf from thevariety LA BU 21. In an aspect, a cured leaf from a tobacco plant,variety, or line comprising a mutant allele of pmt2 comprises a higherleaf grade than a control cured leaf from the variety LA BU 21. In anaspect, a cured leaf from a tobacco plant, variety, or line comprising amutant allele of pmt3 comprises a higher leaf grade than a control curedleaf from the variety LA BU 21. In an aspect, a cured leaf from atobacco plant, variety, or line comprising a mutant allele of pmt4comprises a higher leaf grade than a control cured leaf from the varietyLA BU 21. In another aspect, a cured tobacco leaf from a plant, variety,or line comprising a mutant allele of pmt1a, a mutant allele of pmt1b, amutant allele of pmt2, a mutant allele of pmt3, and a mutant allele ofpmt4 comprises a higher leaf grade than a control cured leaf from thevariety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or lineprovided in any one of Tables 4A to 4E, Table 10, or Table 14 comprisesa higher leaf grade than a control cured leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or linecomprising one or more pint mutations provided in any one of Tables 5Ato 5E and Tables 12A to 12E comprises a higher leaf grade than a controlcured leaf from the variety LA BU 21.

In an aspect, a “reduced mold infection” refers to a reduced area ofinfected leaf. In another aspect, a “reduced mold infection” refers to areduced number of viable mold spores on an infected leaf. Standardmethods of detecting and counting viable mold spores are known andavailable in the art.

In an aspect, a reduced mold infection comprises a reduction of infectedleaf area of at least 1% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area of atleast 2% as compared to a control leaf. In an aspect, a reduced moldinfection comprises a reduction of infected leaf area of at least 3% ascompared to a control leaf. In an aspect, a reduced mold infectioncomprises a reduction of infected leaf area of at least 4% as comparedto a control leaf. In an aspect, a reduced mold infection comprises areduction of infected leaf area of at least 5% as compared to a controlleaf. In an aspect, a reduced mold infection comprises a reduction ofinfected leaf area of at least 10% as compared to a control leaf. In anaspect, a reduced mold infection comprises a reduction of infected leafarea of at least 15% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area of atleast 20% as compared to a control leaf. In an aspect, a reduced moldinfection comprises a reduction of infected leaf area of at least 25% ascompared to a control leaf. In an aspect, a reduced mold infectioncomprises a reduction of infected leaf area of at least 30% as comparedto a control leaf. In an aspect, a reduced mold infection comprises areduction of infected leaf area of at least 35% as compared to a controlleaf. In an aspect, a reduced mold infection comprises a reduction ofinfected leaf area of at least 40% as compared to a control leaf. In anaspect, a reduced mold infection comprises a reduction of infected leafarea of at least 50% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area of atleast 60% as compared to a control leaf. In an aspect, a reduced moldinfection comprises a reduction of infected leaf area of at least 70% ascompared to a control leaf. In an aspect, a reduced mold infectioncomprises a reduction of infected leaf area of at least 75% as comparedto a control leaf. In an aspect, a reduced mold infection comprises areduction of infected leaf area of at least 80% as compared to a controlleaf. In an aspect, a reduced mold infection comprises a reduction ofinfected leaf area of at least 90% as compared to a control leaf. In anaspect, a reduced mold infection comprises a reduction of infected leafarea of at least 95% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area of100% as compared to a control leaf.

In an aspect, a reduced mold infection comprises a reduction of infectedleaf area of between 1% and 100% as compared to a control leaf. In anaspect, a reduced mold infection comprises a reduction of infected leafarea of between 1% and 90% as compared to a control leaf. In an aspect,a reduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 80% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 70% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 60% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 50% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 40% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 30% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 20% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 1% and 10% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 10% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 20% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 30% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 40% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 50% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 60% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 70% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 80% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 90% and 100% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 10% and 75% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 25% and 75% as compared to a control leaf. In an aspect, areduced mold infection comprises a reduction of infected leaf area ofbetween 25% and 50% as compared to a control leaf.

In an aspect, mold infecting cured tobacco is of a genus selected fromthe group consisting of Cladosporium, Penicillium, Alternaria,Aspergillus, and Mucor.

Tobacco material obtained from the tobacco lines, varieties or hybridsof the present disclosure can be used to make tobacco products. As usedherein, “tobacco product” is defined as any product made or derived fromtobacco that is intended for human use or consumption.

Tobacco products provided include, without limitation, cigaretteproducts (e.g., cigarettes and bidi cigarettes), cigar products (e.g.,cigar wrapping tobacco and cigarillos), pipe tobacco products, productsderived from tobacco, tobacco-derived nicotine products, smokelesstobacco products (e.g., moist snuff, dry snuff, and chewing tobacco),films, chewables, tabs, shaped parts, gels, consumable units, insolublematrices, hollow shapes, reconstituted tobacco, expanded tobacco, andthe like. See, e.g., U.S. Patent Publication No. US 2006/0191548.

As used herein, “cigarette” refers a tobacco product having a “rod” and“filler”. The cigarette “rod” includes the cigarette paper, filter, plugwrap (used to contain filtration materials), tipping paper that holdsthe cigarette paper (including the filler) to the filter, and all gluesthat hold these components together. The “filler” includes (1) alltobaccos, including but not limited to reconstituted and expandedtobacco, (2) non-tobacco substitutes (including but not limited toherbs, non-tobacco plant materials and other spices that may accompanytobaccos rolled within the cigarette paper), (3) casings, (4)flavorings, and (5) all other additives (that are mixed into tobaccosand substitutes and rolled into the cigarette).

As used herein, “reconstituted tobacco” refers to a part of tobaccofiller made from tobacco dust and other tobacco scrap material,processed into sheet form and cut into strips to resemble tobacco. Inaddition to the cost savings, reconstituted tobacco is very importantfor its contribution to cigarette taste from processing flavordevelopment using reactions between ammonia and sugars.

As used herein, “expanded tobacco” refers to a part of tobacco fillerwhich is processed through expansion of suitable gases so that thetobacco is “puffed” resulting in reduced density and greater fillingcapacity. It reduces the weight of tobacco used in cigarettes.

Tobacco products derived from plants of the present disclosure alsoinclude cigarettes and other smoking articles, particularly thosesmoking articles including filter elements, where the rod of smokablematerial includes cured tobacco within a tobacco blend. In an aspect, atobacco product of the present disclosure is selected from the groupconsisting of a cigarillo, a non-ventilated recess filter cigarette, avented recess filter cigarette, a cigar, snuff, pipe tobacco, cigartobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookahtobacco, shredded tobacco, and cut tobacco. In another aspect, a tobaccoproduct of the present disclosure is a smokeless tobacco product.Smokeless tobacco products are not combusted and include, but notlimited to, chewing tobacco, moist smokeless tobacco, snus, and drysnuff. Chewing tobacco is coarsely divided tobacco leaf that istypically packaged in a large pouch-like package and used in a plug ortwist. Moist smokeless tobacco is a moist, more finely divided tobaccothat is provided in loose form or in pouch form and is typicallypackaged in round cans and used as a pinch or in a pouch placed betweenan adult tobacco consumer's cheek and gum. Snus is a heat treatedsmokeless tobacco. Dry snuff is finely ground tobacco that is placed inthe mouth or used nasally. In a further aspect, a tobacco product of thepresent disclosure is selected from the group consisting of loose leafchewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. Inyet another aspect, a tobacco product of the present disclosure isselected from the group consisting of an electronically heatedcigarette, an e-cigarette, an electronic vaporing device.

In an aspect, a tobacco product of the present disclosure can be ablended tobacco product. In another aspect, a tobacco product of thepresent disclosure can be a low nicotine tobacco product. In a furtheraspect, a tobacco product of the present disclosure may comprisenornicotine at a level of less than about 3 mg/g. For example, thenornicotine content in such a product can be 3.0 mg/g, 2.5 mg/g, 2.0mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g,1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g,or undetectable.

In an aspect, cured tobacco material or tobacco products providedcomprise an average nicotine or total alkaloid level selected from thegroup consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%,0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9% on a dry weight basis. In anotheraspect, cured tobacco material or tobacco products provided comprise anaverage nicotine or total alkaloid level selected from the groupconsisting of about between 0.01% and 0.02%, between 0.02% and 0.05%,between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%,between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%,between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%,between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%,between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1%and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%,between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%,between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis.In a further aspect, cured tobacco material or tobacco products providedcomprise an average nicotine or total alkaloid level selected from thegroup consisting of about between 0.01% and 0.1%, between 0.02% and0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%,between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis.

The present disclosure also provides methods for breeding tobacco lines,cultivars, or varieties comprising a desirable level of total alkaloidor nicotine, e.g., low nicotine or nicotine free. Breeding can becarried out via any known procedures. DNA fingerprinting, SNP mapping,haplotype mapping or similar technologies may be used in amarker-assisted selection (MAS) breeding program to transfer or breed adesirable trait or allele into a tobacco plant. For example, a breedercan create segregating populations in a F₂ or backcross generation usingF1 hybrid plants or further crossing the F1 hybrid plants with otherdonor plants with an agronomically desirable genotype. Plants in the F₂or backcross generations can be screened for a desired agronomic traitor a desirable chemical profile using one of the techniques known in theart or listed herein. Depending on the expected inheritance pattern orthe MAS technology used, self-pollination of selected plants before eachcycle of backcrossing to aid identification of the desired individualplants can be performed. Backcrossing or other breeding procedure can berepeated until the desired phenotype of the recurrent parent isrecovered. A recurrent parent in the present disclosure can be aflue-cured variety, a Burley variety, a dark air-cured variety, a darkfire-cured variety, or an Oriental variety. Other breeding techniquescan be found, for example, in Wernsman, E. A., and Rufty, R. C. 1987.Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. CropSpecies. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York,N.Y., incorporated herein by reference in their entirety.

Results of a plant breeding program using the tobacco plants describedincludes useful lines, cultivars, varieties, progeny, inbreds, andhybrids of the present disclosure. As used herein, the term “variety”refers to a population of plants that share constant characteristicswhich separate them from other plants of the same species. A variety isoften, although not always, sold commercially. While possessing one ormore distinctive traits, a variety is further characterized by a verysmall overall variation between individuals within that variety. A “pureline” variety may be created by several generations of self-pollinationand selection, or vegetative propagation from a single parent usingtissue or cell culture techniques. A variety can be essentially derivedfrom another line or variety. As defined by the International Conventionfor the Protection of New Varieties of Plants (Dec. 2, 1961, as revisedat Geneva on Nov. 10, 1972; on Oct. 23, 1978; and on Mar. 19, 1991), avariety is “essentially derived” from an initial variety if: a) it ispredominantly derived from the initial variety, or from a variety thatis predominantly derived from the initial variety, while retaining theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety; b) it isclearly distinguishable from the initial variety; and c) except for thedifferences which result from the act of derivation, it conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety. Essentially derived varieties can be obtained, for example, bythe selection of a natural or induced mutant, a somaclonal variant, avariant individual from plants of the initial variety, backcrossing, ortransformation. A first tobacco variety and a second tobacco varietyfrom which the first variety is essentially derived, are considered ashaving essentially identical genetic background. A “line” asdistinguished from a variety most often denotes a group of plants usednon-commercially, for example in plant research. A line typicallydisplays little overall variation between individuals for one or moretraits of interest, although there may be some variation betweenindividuals for other traits.

In an aspect, this disclosure provides a tobacco plant, variety, line,or cell comprising one or more pmt mutations provided in any one ofTables 5A to 5E and Tables 12A to 12E.

In another aspect, this disclosure provides a tobacco plant, variety,line, or cell derived from any tobacco plant, variety, or line providedin any one of Tables 4A to 4E, Table 10, or Table 14.

In an aspect, this disclosure provides the tobacco line 18GH203 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH341 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 17GH1678 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 17GH1680 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 17GH1804 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 17GH1898 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH207 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH342 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH343 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH348 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH349 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH355 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH359 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH64 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH682 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH692 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH697 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH922 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH957 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1808 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1810 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1886 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1888 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1889 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH189 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1893 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1901 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1902 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH3 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH125 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH208 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH403 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH414 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH434 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH436 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH437 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH449 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH706 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH709 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH710 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH716 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH729 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH731 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH752 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH756 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH768 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH771 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH776 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH800 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH818 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH10 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH1004 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH1033 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH132 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH134 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH217 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH456 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH457 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH460 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH465 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH71 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH830 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH831 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH836 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH841 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH974 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH981 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH994 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1905 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH128 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH130 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH131 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH133 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH136 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH216 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH227 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH5 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH6 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH65 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH66 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH69 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH72 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH73 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH74 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH78 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH79 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH8 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH9 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1696 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1717 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1719 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1729 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1736 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1737 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1739 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1740 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1835 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1848 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1849 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1912 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1937 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1940 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1943 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1944 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH1051 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH22 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH34 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH473 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH49 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH50 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH848 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH850 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH851 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1699 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1708 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1722 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1724 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1725 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1845 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1846 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1847 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1911 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1912 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1915 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1918 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1928 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1932 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1933 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1936 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH20 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH28 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH31 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH47 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH51 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH52 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS107 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS106 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS115 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1809-13 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line CS111 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS112 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1678-60 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line CS131 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH709-01 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH709-08 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 18GH414-11 andF₁ or F₂ tobacco plants, or male sterile tobacco plants, derivedtherefrom. In an aspect, this disclosure provides the tobacco line18GH414-19 and F₁ or F₂ tobacco plants, or male sterile tobacco plants,derived therefrom. In an aspect, this disclosure provides the tobaccoline 18GH437-04 and F₁ or F₂ tobacco plants, or male sterile tobaccoplants, derived therefrom. In an aspect, this disclosure provides thetobacco line 18GH437-08 and F₁ or F₂ tobacco plants, or male steriletobacco plants, derived therefrom. In an aspect, this disclosureprovides the tobacco line 18GH437-32 and F₁ or F₂ tobacco plants, ormale sterile tobacco plants, derived therefrom. In an aspect, thisdisclosure provides the tobacco line 18GH437-39 and F₁ or F₂ tobaccoplants, or male sterile tobacco plants, derived therefrom. In an aspect,this disclosure provides the tobacco line 18GH449-26 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH449-33 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 18GH125-48 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line CS102 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line CS103 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 17GH1719-30 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom. Inan aspect, this disclosure provides the tobacco line 17GH1740-36 and F₁or F₂ tobacco plants, or male sterile tobacco plants, derived therefrom.In an aspect, this disclosure provides the tobacco line 17GH1698-22 andF₁ or F₂ tobacco plants, or male sterile tobacco plants, derivedtherefrom. In an aspect, this disclosure provides the tobacco line17GH1700-13 and F₁ or F₂ tobacco plants, or male sterile tobacco plants,derived therefrom. In an aspect, this disclosure provides the tobaccoline 17GH1702-17 and F₁ or F₂ tobacco plants, or male sterile tobaccoplants, derived therefrom. In an aspect, this disclosure provides thetobacco line 17GH1849-01 and F₁ or F₂ tobacco plants, or male steriletobacco plants, derived therefrom. In an aspect, this disclosureprovides the tobacco line 17GH1849-48 and F₁ or F₂ tobacco plants, ormale sterile tobacco plants, derived therefrom. In an aspect, thisdisclosure provides the tobacco line 17GH1737-24 and F₁ or F₂ tobaccoplants, or male sterile tobacco plants, derived therefrom. In an aspect,this disclosure provides the tobacco line CS118 and F₁ or F₂ tobaccoplants, or male sterile tobacco plants, derived therefrom. In an aspect,this disclosure provides the tobacco line CS133 and F₁ or F₂ tobaccoplants, or male sterile tobacco plants, derived therefrom. In an aspect,this disclosure provides the tobacco line CS120 and F₁ or F₂ tobaccoplants, or male sterile tobacco plants, derived therefrom. In an aspect,this disclosure provides the tobacco line 18GH1108-07 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH2162 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS164 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS163 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS146 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS147 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS150 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS151 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS148 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS149 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS152 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS153 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS143 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH2169 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH2171 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS165 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line CS118 and F₁ or F₂tobacco plants, or male sterile tobacco plants, derived therefrom. In anaspect, this disclosure provides the tobacco line 18GH2254-7 and F₁ orF₂ tobacco plants, or male sterile tobacco plants, derived therefrom.

In an aspect, the present disclosure provides a method of introgressinga low nicotine trait into a tobacco variety, the method comprising: (a)crossing a first tobacco variety comprising a low nicotine trait with asecond tobacco variety without the low nicotine trait to produce one ormore progeny tobacco plants; (b) genotyping the one or more progenytobacco plants for a pmt mutant allele selected from those listed inTables 4A to 4E, Tables 5A to 5E, Table 10, and Tables 12A to 12E; and(c) selecting a progeny tobacco plant comprising the pmt mutant allele.In another aspect, these methods further comprise backcrossing theselected progeny tobacco plant with the second tobacco variety. In afurther aspect, these methods further comprise: (d) crossing theselected progeny plant with itself or with the second tobacco variety toproduce one or more further progeny tobacco plants; and (e) selecting afurther progeny tobacco plant comprising a low nicotine trait. In anaspect, the step (e) of selecting comprises marker-assisted selection.In an aspect, these methods produce a single gene conversion comprisinga low nicotine trait. In an aspect, these methods produce a single geneconversion comprising a pmt mutant allele. In an aspect, the secondtobacco variety is an elite variety. In another aspect, the genotypingstep of these methods involve one or more molecular marker assays. Inanother aspect, the genotyping may involve a polymorphic markercomprising a polymorphism selected from the group consisting of singlenucleotide polymorphisms (SNPs), insertions or deletions in DNA sequence(Indels), simple sequence repeats of DNA sequence (SSRs), a restrictionfragment length polymorphism (RFLP), and a tag SNP.

As used herein, “locus” is a chromosomal locus or region where apolymorphic nucleic acid, trait determinant, gene, or marker is located.A “locus” can be shared by two homologous chromosomes to refer to theircorresponding locus or region. As used herein, “allele” refers to analternative nucleic acid sequence of a gene or at a particular locus(e.g., a nucleic acid sequence of a gene or locus that is different thanother alleles for the same gene or locus). Such an allele can beconsidered (i) wild-type or (ii) mutant if one or more mutations oredits are present in the nucleic acid sequence of the mutant allelerelative to the wild-type allele. A mutant allele for a gene may have areduced or eliminated activity or expression level for the gene relativeto the wild-type allele. For diploid organisms such as tobacco, a firstallele can occur on one chromosome, and a second allele can occur at thesame locus on a second homologous chromosome. If one allele at a locuson one chromosome of a plant is a mutant allele and the othercorresponding allele on the homologous chromosome of the plant iswild-type, then the plant is described as being heterozygous for themutant allele. However, if both alleles at a locus are mutant alleles,then the plant is described as being homozygous for the mutant alleles.A plant homozygous for mutant alleles at a locus may comprise the samemutant allele or different mutant alleles if heteroallelic or biallelic.

As used herein, “introgression” or “introgress” refers to thetransmission of a desired allele of a genetic locus from one geneticbackground to another.

As used herein, “crossed” or “cross” means to produce progeny viafertilization (e.g. cells, seeds or plants) and includes crosses betweenplants (sexual) and self-fertilization (selfing).

As used herein, “backcross” and “backcrossing” refer to the processwhereby a progeny plant is repeatedly crossed back to one of itsparents. In a backcrossing scheme, the “donor” parent refers to theparental plant with the desired gene or locus to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. The initial cross gives rise to the F1generation. The term “BC1” refers to the second use of the recurrentparent, “BC2” refers to the third use of the recurrent parent, and soon. In an aspect, a backcross is performed repeatedly, with a progenyindividual of each successive backcross generation being itselfbackcrossed to the same parental genotype.

As used herein, “single gene converted” or “single gene conversion”refers to plants that are developed using a plant breeding techniqueknown as backcrossing, or via genetic engineering, where essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety via the backcrossing technique or via genetic engineering.

As used herein, “elite variety” means any variety that has resulted frombreeding and selection for superior agronomic performance.

As used herein, “selecting” or “selection” in the context ofmarker-assisted selection or breeding refer to the act of picking orchoosing desired individuals, normally from a population, based oncertain pre-determined criteria.

As used herein, the term “trait” refers to one or more detectablecharacteristics of a cell or organism which can be influenced bygenotype. The phenotype can be observable to the naked eye, or by anyother means of evaluation known in the art, e.g., microscopy,biochemical analysis, genomic analysis, an assay for a particulardisease tolerance, etc. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, e.g., a “single genetrait.” In other cases, a phenotype is the result of several genes.

As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.,measurement of at least one phenotype (such as seed color, flower color,or other visually detectable trait), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies, andnucleic acid sequencing technologies, etc.

As used herein, “marker assisted selection” (MAS) is a process by whichphenotypes are selected based on marker genotypes. “Marker assistedselection breeding” refers to the process of selecting a desired traitor traits in a plant or plants by detecting one or more nucleic acidsfrom the plant, where the nucleic acid is linked to the desired trait,and then selecting the plant or germplasm possessing those one or morenucleic acids.

As used herein, “polymorphism” means the presence of one or morevariations in a population. A polymorphism may manifest as a variationin the nucleotide sequence of a nucleic acid or as a variation in theamino acid sequence of a protein. Polymorphisms include the presence ofone or more variations of a nucleic acid sequence or nucleic acidfeature at one or more loci in a population of one or more individuals.The variation may comprise but is not limited to one or more nucleotidebase changes, the insertion of one or more nucleotides or the deletionof one or more nucleotides. A polymorphism may arise from randomprocesses in nucleic acid replication, through mutagenesis, as a resultof mobile genomic elements, from copy number variation and during theprocess of meiosis, such as unequal crossing over, genome duplicationand chromosome breaks and fusions. The variation can be commonly foundor may exist at low frequency within a population, the former havinggreater utility in general plant breeding and the latter may beassociated with rare but important phenotypic variation. Usefulpolymorphisms may include single nucleotide polymorphisms (SNPs),insertions or deletions in DNA sequence (Indels), simple sequencerepeats of DNA sequence (SSRs), a restriction fragment lengthpolymorphism (RFLP), and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a RNA-derived sequence, a promoter, a 5′untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a tolerance locus, a satellite marker, a transgene,mRNA, ds mRNA, a transcriptional profile, and a methylation pattern mayalso comprise polymorphisms. In addition, the presence, absence, orvariation in copy number of the preceding may comprise polymorphisms.

As used herein, “SNP” or “single nucleotide polymorphism” means asequence variation that occurs when a single nucleotide (A, T, C, or G)in the genome sequence is altered or variable. “SNP markers” exist whenSNPs are mapped to sites on the genome.

As used herein, “marker” or “molecular marker” or “marker locus” is aterm used to denote a nucleic acid or amino acid sequence that issufficiently unique to characterize a specific locus on the genome. Anydetectable polymorphic trait can be used as a marker so long as it isinherited differentially and exhibits linkage disequilibrium with aphenotypic trait of interest. Each marker is therefore an indicator of aspecific segment of DNA, having a unique nucleotide sequence. The mappositions provide a measure of the relative positions of particularmarkers with respect to one another. When a trait is stated to be linkedto a given marker it will be understood that the actual DNA segmentwhose sequence affects the trait generally co-segregates with themarker. More precise and definite localization of a trait can beobtained if markers are identified on both sides of the trait. Bymeasuring the appearance of the marker(s) in progeny of crosses, theexistence of the trait can be detected by relatively simple moleculartests without actually evaluating the appearance of the trait itself,which can be difficult and time-consuming because the actual evaluationof the trait requires growing plants to a stage and/or underenvironmental conditions where the trait can be expressed.

It is understood that any tobacco plant of the present disclosure canfurther comprise additional agronomically desirable traits, for example,by transformation with a genetic construct or transgene using atechnique known in the art. Without limitation, an example of a desiredtrait is herbicide resistance, pest resistance, disease resistance; highyield; high grade index value; curability; curing quality; mechanicalharvestability; holding ability; leaf quality; height, plant maturation(e.g., early maturing, early to medium maturing, medium maturing, mediumto late maturing, or late maturing); stalk size (e.g., a small, medium,or a large stalk); or leaf number per plant (e.g., a small (e.g., 5-10leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number ofleaves), or any combination. In an aspect, low-nicotine or nicotine-freetobacco plants or seeds disclosed comprise one or more transgenesexpressing one or more insecticidal proteins, such as, for example, acrystal protein of Bacillus thuringiensis or a vegetative insecticidalprotein from Bacillus cereus, such as VIP3 (see, for example, Estruch etal. (1997) Nat. Biotechnol. 15:137). In another aspect, tobacco plantsfurther comprise an introgressed trait conferring resistance to brownstem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S.Pat. No. 5,491,081).

The present disclosure also provides pmt mutant tobacco plantscomprising an altered nicotine or total alkaloid level but having ayield comparable to the yield of corresponding initial tobacco plantswithout such a nicotine level alternation. In an aspect, a pmt mutantvariety provides a yield selected from the group consisting of aboutbetween 1200 and 3500, between 1300 and 3400, between 1400 and 3300,between 1500 and 3200, between 1600 and 3100, between 1700 and 3000,between 1800 and 2900, between 1900 and 2800, between 2000 and 2700,between 2100 and 2600, between 2200 and 2500, and between 2300 and 2400lbs/acre. In another aspect, a pmt mutant tobacco variety provides ayield selected from the group consisting of about between 1200 and 3500,between 1300 and 3500, between 1400 and 3500, between 1500 and 3500,between 1600 and 3500, between 1700 and 3500, between 1800 and 3500,between 1900 and 3500, between 2000 and 3500, between 2100 and 3500,between 2200 and 3500, between 2300 and 3500, between 2400 and 3500,between 2500 and 3500, between 2600 and 3500, between 2700 and 3500,between 2800 and 3500, between 2900 and 3500, between 3000 and 3500, andbetween 3100 and 3500 lbs/acre. In a further aspect, pmt mutant tobaccoplants provide a yield between 65% and 130%, between 70% and 130%,between 75% and 130%, between 80% and 130%, between 85% and 130%,between 90% and 130%, between 95% and 130%, between 100% and 130%,between 105% and 130%, between 110% and 130%, between 115% and 130%, orbetween 120% and 130% of the yield of a control plant having essentiallyidentical genetic background except for pmt mutation(s). In a furtheraspect, pmt mutant tobacco plants provide a yield between 70% and 125%,between 75% and 120%, between 80% and 115%, between 85% and 110%, orbetween 90% and 100% of the yield of a control plant having essentiallyidentical genetic background except for pmt mutations.

In an aspect, a tobacco plant disclosed (e.g., a low-nicotine,nicotine-free, or low-alkaloid tobacco variety) comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) without substantially impacting a trait selected from thegroup consisting of yield, ripening and senescence, susceptibility toinsect herbivory, polyamine content after topping, chlorophyll level,mesophyll cell number per unit leaf area, and end-product quality aftercuring.

In an aspect, a tobacco plant disclosed comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a trait substantially comparable toan unmodified control plant, where the trait is selected from the groupconsisting of yield, ripening and senescence, susceptibility to insectherbivory, polyamine content after topping, chlorophyll level, mesophyllcell number per unit leaf area, and end-product quality after curing.

In an aspect, a tobacco plant disclosed comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a yield which is more than 80%, morethan 85%, more than 90%, more than 95%, more than 100%, more than 105%,more than 110%, more than 115%, more than 120%, more than 125%, morethan 130%, more than 135%, or more than 140% relative to the yield of anunmodified control plant. In an aspect, a tobacco plant disclosedcomprises a modification conferring a desired trait (e.g., low-nicotine,nicotine-free, or low-alkaloid) and further comprises a yield which isbetween 70% and 140%, between 75% and 135%, between 80% and 130%,between 85% and 125%, between 90% and 120%, between 95% and 115%, orbetween 100% and 110% relative to the yield of an unmodified controlplant. In an aspect, a tobacco plant disclosed comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a yield which is between 70% and80%, between 75% and 85%, between 80% and 90%, between 85% and 95%,between 90% and 100%, between 95% and 105%, between 105% and 115%,between 110% and 120%, between 115% to 125%, between 120% and 130%,between 125 and 135%, or between 130% and 140% relative to the yield ofan unmodified control plant.

In an aspect, a low-nicotine or nicotine-free tobacco variety disclosedis adapted for machine harvesting. In another aspect, a low-nicotine ornicotine-free tobacco variety disclosed is harvested mechanically.

In an aspect, tobacco plants provided are hybrid plants. Hybrids can beproduced by preventing self-pollination of female parent plants (e.g.,seed parents) of a first variety, permitting pollen from male parentplants of a second variety to fertilize the female parent plants, andallowing F1 hybrid seeds to form on the female plants. Self-pollinationof female plants can be prevented by emasculating the flowers at anearly stage of flower development. Alternatively, pollen formation canbe prevented on the female parent plants using a form of male sterility.For example, male sterility can be produced by male sterility (MS), ortransgenic male sterility where a transgene inhibits microsporogenesisand/or pollen formation, or self-incompatibility. Female parent plantscontaining MS are particularly useful. In aspects in which the femaleparent plants are MS, pollen may be harvested from male fertile plantsand applied manually to the stigmas of MS female parent plants, and theresulting F1 seed is harvested.

Plants can be used to form single-cross tobacco F1 hybrids. Pollen froma male parent plant is manually transferred to an emasculated femaleparent plant or a female parent plant that is male sterile to form F1seed. Alternatively, three-way crosses can be carried out where asingle-cross F1 hybrid is used as a female parent and is crossed with adifferent male parent. As another alternative, double-cross hybrids canbe created where the F1 progeny of two different single-crosses arethemselves crossed. Self-incompatibility can be used to particularadvantage to prevent self-pollination of female parents when forming adouble-cross hybrid.

In an aspect, a low-nicotine or nicotine-free tobacco variety is malesterile. In another aspect, a low-nicotine or nicotine-free tobaccovariety is cytoplasmic male sterile. Male sterile tobacco plants may beproduced by any method known in the art. Methods of producing malesterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987.Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. CropSpecies. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York,N.Y. 761 pp.

In an aspect, this disclosure provides a male sterile tobacco plant,variety, or line comprising one or more pmt mutations provided in anyone of Tables 5A to 5E and Tables 12A to 12E.

In another aspect, this disclosure provides a male sterile tobaccoplant, variety, or line derived from any tobacco plant, variety, or lineprovided in any one of Tables 4A to 4E, Table 10, or Table 14.

In an aspect, this disclosure provides the male sterile line dCS11. Inanother aspect, this disclosure provides the male sterile line dCS12. Inanother aspect, this disclosure provides the male sterile line dCS13. Inanother aspect, this disclosure provides the male sterile line dCS14. Inanother aspect, this disclosure provides the male sterile line dCS15. Inanother aspect, this disclosure provides the male sterile line dCS16. Inanother aspect, this disclosure provides the male sterile line dCS17. Inanother aspect, this disclosure provides the male sterile line dCS18. Inanother aspect, this disclosure provides the male sterile line dS697.

In a further aspect, tobacco parts provided include, but are not limitedto, a leaf, a stem, a root, a seed, a flower, pollen, an anther, anovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod,an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, acell, and a protoplast. In an aspect, tobacco part provided does notinclude seed. In an aspect, this disclosure provides tobacco plantcells, tissues, and organs that are not reproductive material and do notmediate the natural reproduction of the plant. In another aspect, thisdisclosure also provides tobacco plant cells, tissues, and organs thatare reproductive material and mediate the natural reproduction of theplant. In another aspect, this disclosure provides tobacco plant cells,tissues, and organs that cannot maintain themselves via photosynthesis.In another aspect, this disclosure provides somatic tobacco plant cells.Somatic cells, contrary to germline cells, do not mediate plantreproduction.

Cells, tissues and organs can be from seed, fruit, leaf, cotyledon,hypocotyl, meristem, embryos, endosperm, root, shoot, stem, pod, flower,infloresence, stalk, pedicel, style, stigma, receptacle, petal, sepal,pollen, anther, filament, ovary, ovule, pericarp, phloem, vasculartissue. In another aspect, this disclosure provides a tobacco plantchloroplast. In a further aspect, this disclosure provides epidermalcells, stomata cell, leaf or root hairs, a storage root, or a tuber. Inanother aspect, this disclosure provides a tobacco protoplast.

Skilled artisans understand that tobacco plants naturally reproduce viaseeds, not via asexual reproduction or vegetative propagation. In anaspect, this disclosure provides tobacco endosperm. In another aspect,this disclosure provides tobacco endosperm cells. In a further aspect,this disclosure provides a male or female sterile tobacco plant, whichcannot reproduce without human intervention.

In an aspect, the present disclosure provides a nucleic acid moleculecomprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70% 75% 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto a sequence selected from the group consisting of SEQ ID NOs: 1 to 10,and fragments thereof. In an aspect, the present disclosure provides apolypeptide or protein comprising at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 11 to 15.

As used herein, the term “sequence identity” or “identity” in thecontext of two polynucleotides or polypeptide sequences makes referenceto the residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution.

The present disclosure further provides a method manufacturing a tobaccoproduct comprising tobacco material from tobacco plants disclosed. In anaspect, methods comprise conditioning aged tobacco material made fromtobacco plants to increase its moisture content from between about 12.5%and about 13.5% to about 21%, blending the conditioned tobacco materialto produce a desirable blend. In an aspect, the method of manufacturinga tobacco product further comprises casing or flavoring the blend.Generally, during the casing process, casing or sauce materials areadded to blends to enhance their quality by balancing the chemicalcomposition and to develop certain desired flavor characteristics.Further details for the casing process can be found in TobaccoProduction, Chemistry and Technology, Edited by L. Davis and M. Nielsen,Blackwell Science, 1999.

Tobacco material provided can be also processed using methods including,but not limited to, heat treatment (e.g., cooking, toasting), flavoring,enzyme treatment, expansion and/or curing. Both fermented andnon-fermented tobaccos can be processed using these techniques. Examplesof suitable processed tobaccos include dark air-cured, dark fire cured,burley, flue cured, and cigar filler or wrapper, as well as the productsfrom the whole leaf stemming operation. In an aspect, tobacco fibersinclude up to 70% dark tobacco on a fresh weight basis. For example,tobacco can be conditioned by heating, sweating and/or pasteurizingsteps as described in U.S. Publication Nos. 2004/0118422 or2005/0178398.

Tobacco material provided can be subject to fermentation. Fermentingtypically is characterized by high initial moisture content, heatgeneration, and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat.Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition tomodifying the aroma of the leaf, fermentation can change either or boththe color and texture of a leaf. Also during the fermentation process,evolution gases can be produced, oxygen can be taken up, the pH canchange, and the amount of water retained can change. See, for example,U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cured, or cured and fermented tobacco can befurther processed (e.g., cut, expanded, blended, milled or comminuted)prior to incorporation into the oral product. The tobacco, in somecases, is long cut fermented cured moist tobacco having an ovenvolatiles content of between 48 and 50 weight percent prior to mixingwith the copolymer and optionally flavorants and other additives.

In an aspect, tobacco material provided can be processed to a desiredsize. In an aspect, tobacco fibers can be processed to have an averagefiber size of less than 200 micrometers. In an aspect, tobacco fibersare between 75 and 125 micrometers. In another aspect, tobacco fibersare processed to have a size of 75 micrometers or less. In an aspect,tobacco fibers include long cut tobacco, which can be cut or shreddedinto widths of about 10 cuts/inch up to about 110 cuts/inch and lengthsof about 0.1 inches up to about 1 inch. Double cut tobacco fibers canhave a range of particle sizes such that about 70% of the double cuttobacco fibers falls between the mesh sizes of −20 mesh and 80 mesh.

Tobacco material provided can be processed to have a total ovenvolatiles content of about 10% by weight or greater; about 20% by weightor greater; about 40% by weight or greater; about 15% by weight to about25% by weight; about 20% by weight to about 30% by weight; about 30% byweight to about 50% by weight; about 45% by weight to about 65% byweight; or about 50% by weight to about 60% by weight. Those of skill inthe art will appreciate that “moist” tobacco typically refers to tobaccothat has an oven volatiles content of between about 40% by weight andabout 60% by weight (e.g., about 45% by weight to about 55% by weight,or about 50% by weight). As used herein, “oven volatiles” are determinedby calculating the percentage of weight loss for a sample after dryingthe sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours.The oral product can have a different overall oven volatiles contentthan the oven volatiles content of the tobacco fibers used to make theoral product. The processing steps described can reduce or increase theoven volatiles content.

Having now generally described the disclosure, the same will be morereadily understood through reference to the following examples that areprovided by way of illustration, and are not intended to be limiting ofthe present disclosure, unless specified.

EXAMPLES Example 1: Expression Profiling of Five PMT Genes

Nicotine biosynthesis starts with conversion of polyamine putrescine toN-methylputrescine by the enzyme putrescine N-methyl transferase (PMT).This is a step that commits precursor metabolites to nicotinebiosynthesis. Genes encoding PMT (PMT1a, PMT1b, PMT2, PMT3 and PMT4) arepresent in the tobacco (Nicotiana tabacum) genome. Table 1A listsgenomic DNA sequences, cDNA sequences, and protein sequences of five PMTgenes. Tables 1B and 1C provide sequence identities among five PMTgenes. Pooled expression levels from before topping to harvest providesupport that, without being limited by any particular theory, PMT1a andPMT3 represent two major PMT genes (FIG. 1).

TABLE 1A Sequences of five tobacco PMT genes. Genomic DNA Sequence(including regions such as promoter, 5′ UTR, cDNA Protein introns, 3′UTR, and Sequence Sequence terminator) (SEQ ID (SEQ ID (SEQ ID Gene NameNo.) No.) No.) PMT1b 1 6 11 PMT1a 2 7 12 PMT2 3 8 13 PMT3 4 9 14 PMT4 510 15

TABLE 1B cDNA sequence identity among five tobacco PMT genes determinedby Clustal2.1. cDNA % identity PMT1a PMT1b PMT2 PMT3 PMT4 PMT1a 100PMT1b 98.85 100 PMT2 91.81 91.71 100 PMT3 93.71 93.53 91.79 100 PMT494.24 94.06 92.75 94.59 100

TABLE 1C Protein sequence identity among five tobacco PMT genesdetermined by Clustal2.1. Protein % identity PMT1a PMT1b PMT2 PMT3 PMT4PMT1a 100 PMT1b 98.4 100 PMT2 95.42 95.75 100 PMT3 97.48 97.76 96.23 100PMT4 96.27 96.8 96.32 97.63 100

TABLE 1D PMT1b genomic sequence (SEQ ID No. 1) annotation. Elementlocation 5′ sequence   1 . . . 1000 exon 1 1001 . . . 1292 intron 1 1293. . . 1464 exon 2 1465 . . . 1541 intron 2 1542 . . . 1623 exon 3 1624 .. . 1851 intron 3 1852 . . . 1971 exon 4 1972 . . . 2044 intron 4 2045 .. . 2143 exon 5 2144 . . . 2215 intron 5 2216 . . . 2333 exon 6 2334 . .. 2529 intron 6 2530 . . . 3033 exon 7 3034 . . . 3166 intron 7 3167 . .. 3260 exon 8 3261 . . . 3317 3′ sequence 3318 . . . 4317

TABLE 1E PMT1b genomic sequence (SEQ ID No. 2) annotation. Elementlocation 5′ sequence   1 . . . 1000 exon 1 1001 . . . 1294 intron 1 1295. . . 1422 exon 2 1423 . . . 1497 intron 2 1498 . . . 1579 exon 3 1580 .. . 1810 intron 3 1811 . . . 1932 exon 4 1933 . . . 2003 intron 4 2004 .. . 2102 exon 5 2103 . . . 2175 intron 5 2176 . . . 2293 exon 6 2294 . .. 2487 intron 6 2488 . . . 2925 exon 7 2926 . . . 3058 intron 7 3059 . .. 3153 exon 8 3154 . . . 3210 3′ sequence 3211 . . . 4210

TABLE 1F PMT2 genomic sequence (SEQ ID No. 3) annotation. Elementlocation 5′ sequence  1 . . . 792 exon 1  793 . . . 1020 intron 1 1021 .. . 1201 exon 2 1202 . . . 1276 intron 2 1277 . . . 1358 exon 3 1359 . .. 1589 intron 3 1590 . . . 1694 exon 4 1695 . . . 1765 intron 4 1766 . .. 1875 exon 5 1876 . . . 1948 intron 5 1949 . . . 2037 exon 6 2038 . . .2231 intron 6 2232 . . . 2397 exon 7 2398 . . . 2530 intron 7 2531 . . .2629 exon 8 2630 . . . 2686 3′ sequence 2687 . . . 3686

TABLE 1G PMT3 genomic sequence (SEQ ID No. 4) annotation. Elementlocation 5′ sequence   1 . . . 1000 exon 1 1001 . . . 1312 intron 1 1313. . . 1562 exon 2 1563 . . . 1637 intron 2 1638 . . . 1731 exon 3 1732 .. . 1962 intron 3 1963 . . . 2050 exon 4 2051 . . . 2121 intron 4 2122 .. . 2230 exon 5 2231 . . . 2303 intron 5 2304 . . . 2397 exon 6 2398 . .. 2591 intron 6 2592 . . . 2750 exon 7 2751 . . . 2883 intron 7 2884 . .. 2978 exon 8 2979 . . . 3035 3′ sequence 3036 . . . 4035

TABLE 1H PMT4 genomic sequence (SEQ ID No. 5) annotation. Elementlocation 5′ sequence   1 . . . 1000 exon 1 1001 . . . 1426 intron 1 1427. . . 1609 exon 2 1610 . . . 1684 intron 2 1685 . . . 1766 exon 3 1767 .. . 1997 intron 3 1998 . . . 2112 exon 4 2113 . . . 2183 intron 4 2184 .. . 2290 exon 5 2291 . . . 2363 intron 5 2364 . . . 2452 exon 6 2453 . .. 2646 intron 6 2647 . . . 3146 exon 7 3147 . . . 3279 intron 7 3280 . .. 3374 exon 8 3375 . . . 3431 3′ sequence 3432 . . . 4431

Example 2: PMT Genome Editing and Tobacco Line Development

PMT knockout mutants are produced by editing various PMT genes. Tobaccoprotoplasts are transfected using polyethylene glycol (PEG) withplasmids encoding a genome editing technology 1 (GET 1) protein or agenome editing technology (GET) 2 protein and specific guide RNAs(gRNAs) targeting PMT genes at desired positions. Table 2 lists gRNAsequences used for PMT editing. Some gRNAs (e.g., Nos. 6 and 7) arepooled together for targeting multiple PMT genes in a singletransfection.

Transfected protoplasts are then immobilized in 1% agarose bead andsubjected to tissue culture. When calli grow up to ˜1 mm in diameter,they are spread on TOM2 plates. Calli are screened for insertions ordeletions (indels) at the target positions using fragment analysis.Candidates, showing size shifts compared to wildtype control, areselected for further culture and the consequent shoots are tested byfragment analysis again to confirm the presence of indels. Rooted shootsare potted and sequenced for the target positions to determine the exactsequences deleted. Young leaf from each plant is harvested and PCRamplified for PMT fragments using phirekit. PMT Libraries for each lineis indexed and 384 lines are pooled and sequenced using Miseq.

SNP analysis is carried out to determine both the exact edited pmtmutant allele sequences and the zygosity state at each PMT gene locus.Table 3 provides the zygosity information of representative editedplants. Tables 4A to 4E provide indels sequence information in eachedited line of various tobacco varieties (e.g., K326, TN90, NLM,oriental). Tables 5A to 5E provide genomic sequences of about 40nucleotides from each pmt mutant allele with the edited site in themiddle of the genomic sequence (e.g., 20 nucleotides on each side of thedeleted or inserted sequence site).

TABLE 2 gRNA sequences used in 2 genome editing technologies and theirtarget genes. ″Y″represents that a gRNA targets that PMT gene, while ″—″represents that a gRNA does not target that PMT gene. Genome EditinggRNA Technology Target genes No. (GET) gRNA sequence PMT1b PMT1a PMT2PMT3 PMT4 1 GET 1 CCCATGAACGGCCACCAAAA Y Y — — — (SEQ ID NO: 16) 2 GET 1GGCACTTCCAAACACCAAAA Y Y Y Y Y (SEQ ID NO: 17) 3 GET 1GTTGTTCGGATGTCCCATTC Y Y — — — (SEQ ID NO: 18) 4 GET 1CTAAACTCTGAAAACCAACC Y — Y Y — (SEQ ID NO: 19) 5 GET 1TTTCAGAGTTTAGCGCATTA Y Y Y Y Y (SEQ ID NO: 20) 6 GET 2GATGGAGCAATTCAACATACAGA Y Y — — — (SEQ ID NO: 21) 7 GET 2GATGGAGCAATTCAACACACAGA — — Y Y Y (SEQ ID NO: 22)

TABLE 3 Zygosity of individual PMT genic locus in selected pmt mutantsin various background produced by genome editing using GET2. Number one(1) represents homozygous for a single mutant allele. Numbers 2 to 5represent a heteroallelic combination having 2 to 5 Indels. Hyphensindicate no data. Detailed genotype information is shown in Tables 4A to4D. Variety Line PMT1b PMT1a PMT2 PMT3 PMT4 Basma 18GH203 1 2 2 2 1Basma 18GH341 1 2 2 2 1 K326 17GH1678 2 2 1 1 2 K326 17GH1680 1 2 1 1 1K326 17GH1804 1 2 1 1 1 K326 17GH1898 1 2 1 1 1 K326 18GH207 1 2 1 1 1K326 18GH342 1 2 1 1 1 K326 18GH343 1 2 1 1 1 K326 18GH348 1 1 1 1 1K326 18GH349 1 1 1 1 1 K326 18GH355 1 2 2 3 2 K326 18GH359 1 2 1 1 1K326 18GH64 2 1 2 1 1 K326 18GH682 1 2 2 2 2 K326 18GH692 1 2 2 2 1 K32618GH697 1 1 1 1 1 K326 18GH922 1 1 1 1 1 K326 18GH957 1 1 2 1 1 K32617GH1808 1 2 1 2 1 K326 17GH1810 1 1 1 2 2 K326 17GH1886 — — 2 1 — K32617GH1888 — 1 1 — — K326 17GH1889 — 1 1 — — K326 17GH1892 3 1 2 2 — K32617GH1893 1 1 1 1 1 K326 17GH1901 1 1 1 2 2 K326 17GH1902 1 1 1 2 2 K32618GH3 — 1 1 1 — Katerini 18GH125 2 2 1 2 1 Katerini 18GH208 2 1 1 2 1Katerini 18GH403 1 1 1 1 — Katerini 18GH414 2 1 1 1 1 Katerini 18GH434 21 1 2 1 Katerini 18GH436 2 1 1 4 1 Katerini 18GH437 1 2 1 2 2 Katerini18GH449 2 2 1 1 1 Katerini 18GH706 2 1 1 2 1 Katerini 18GH709 2 2 1 1 1Katerini 18GH710 1 1 2 2 1 Katerini 18GH716 2 2 1 2 2 Katerini 18GH729 11 2 1 1 Katerini 18GH731 1 1 1 2 1 Katerini 18GH752 2 1 1 1 1 Katerini18GH756 1 1 1 2 2 Katerini 18GH768 1 1 1 1 2 Katerini 18GH771 1 1 2 2 1Katerini 18GH776 2 2 1 1 2 Katerini 18GH800 2 2 1 1 2 Katerini 18GH818 11 1 2 — NLMz 18GH10 1 1 1 1 1 NLMz 18GH1004 2 1 1 2 2 NLMz 18GH1033 2 11 2 2 NLMz 18GH132 1 2 2 3 1 NLMz 18GH134 1 2 1 1 1 NLMz 18GH217 1 2 2 12 NLMz 18GH456 2 1 1 1 1 NLMz 18GH457 1 1 1 1 1 NLMz 18GH460 1 2 3 1 1NLMz 18GH465 2 1 1 2 2 NLMz 18GH71 1 1 1 1 1 NLMz 18GH830 1 1 1 1 1 NLMz18GH831 1 2 1 — 1 NLMz 18GH836 1 1 1 1 1 NLMz 18GH841 2 2 1 — 1 NLMz18GH974 2 1 2 2 1 NLMz 18GH981 1 1 2 2 2 NLMz 18GH994 2 1 1 2 2 NLMz17GH1905 1 2 1 2 2 NLMz 18GH128 — 2 2 — 1 NLMz 18GH130 2 2 1 1 1 NLMz18GH131 1 3 2 — 1 NLMz 18GH133 2 3 2 — 1 NLMz 18GH136 — — 1 — — NLMz18GH216 2 2 1 1 2 NLMz 18GH227 1 1 1 — 1 NLMz 18GH5 1 2 1 2 2 NLMz 18GH61 2 3 1 1 NLMz 18GH65 2 2 2 2 1 NLMz 18GH66 1 2 1 2 2 NLMz 18GH69 — 1 —2 1 NLMz 18GH72 2 2 2 2 1 NLMz 18GH73 — 2 2 — 1 NLMz 18GH74 — 1 — — —NLMz 18GH78 1 1 1 3 2 NLMz 18GH79 — 2 2 — 1 NLMz 18GH8 1 2 2 1 2 NLMz18GH9 1 2 1 — 1 TN90 17GH1696 1 1 1 1 1 TN90 17GH1717 1 2 2 2 1 TN9017GH1719 1 2 1 1 1 TN90 17GH1729 2 1 1 1 1 TN90 17GH1736 1 1 2 1 1 TN9017GH1737 1 2 2 2 1 TN90 17GH1739 1 1 1 1 2 TN90 17GH1740 1 2 1 2 1 TN9017GH1835 2 2 2 1 1 TN90 17GH1848 1 2 1 1 1 TN90 17GH1849 1 1 1 2 2 TN9017GH1912 1 2 1 1 1 TN90 17GH1937 1 2 1 2 1 TN90 17GH1940 1 2 1 1 1 TN9017GH1943 1 1 1 1 2 TN90 17GH1944 1 1 1 1 2 TN90 18GH1051 2 2 2 1 2 TN9018GH22 1 2 1 2 1 TN90 18GH34 1 1 1 1 2 TN90 18GH473 1 1 1 2 2 TN9018GH49 1 1 1 1 1 TN90 18GH50 2 1 1 1 1 TN90 18GH848 2 2 2 1 2 TN9018GH850 1 2 1 2 2 TN90 18GH851 1 2 1 2 2 TN90 17GH1699 3 2 2 2 1 TN9017GH1708 1 3 1 2 — TN90 17GH1722 2 1 2 2 1 TN90 17GH1724 2 1 1 2 1 TN9017GH1725 2 1 1 2 1 TN90 17GH1845 2 2 1 2 2 TN90 17GH1846 2 1 2 2 1 TN9017GH1847 2 1 2 2 1 TN90 17GH1911 1 2 1 1 1 TN90 17GH1912 1 2 1 1 1 TN9017GH1915 — 1 — 1 — TN90 17GH1918 2 2 1 1 5 TN90 17GH1928 2 2 — 2 1 TN9017GH1932 2 2 — — 1 TN90 17GH1933 2 2 2 5 1 TN90 17GH1936 2 2 2 1 2 TN9018GH20 — 1 2 1 2 TN90 18GH28 2 1 2 1 2 TN90 18GH31 1 3 1 1 1 TN90 18GH471 3 1 1 1 TN90 18GH51 — — — 1 — TN90 18GH52 — — — 1 —

TABLE 4AMutant pmt alleles in K326 produced by genome editing using GET2. The position of each edited site (e.g.,, indels) is relative to thenucleotide number on the corresponding cDNA sequence of each PMT gene. For example, line 17GH1678 has bi-allelic mutations in PMT1b.One of the two alleles has a four-nucleotide deletion which corresponds to nucleotides 416 to 419 of the PMT1b cDNA sequence. The other allele has a two-nucleotide deletion which corresponds to nucleotides 418 to 419 of the PMT1b cDNA sequence. SEQ ID Numbers are assignedand shown for sequences of more than 10 nucleotides. PMT1b PMT1a PMT2PMT3 PMT4 Deleted Deleted Deleted Deleted Deleted VARIETY LINE Positionsequence Position sequence Position sequence Position sequence Positionsequence K326 17GH 416..419 ATAC 415..421 CATACAG 348..349 AC 432..435ACAC 547..551 CACAC 1678 418..419 AC 417..420 TACA 548..551 ACAC K32617GH 414..417 ACAT 414..417 ACAT 348..349 AC 432..435 ACAC 546..547 AC1680 416..417 AT K326 17GH 414..417 ACAT 411..420 TCAACAT 348..349 AC433..437 CACAC 548..552 ACACA 1804 ACA (379) 417..420 TACA K326 17GH414..417 ACAT 411..420 TCAACAT 348..349 AC 433..437 CACAC 548..552 ACACA1898 ACA (379) 417..420 TACA K326 18GH 414..417 ACAT 415..415 C 348..351ACAC 432..433 AC 546..549 ACAC 207 417..417 T K326 18GH 414..417 ACAT414..417 ACAT 348..349 AC 432..435 ACAC 546..547 AC 343 416..417 AT K32618GH 414..417 ACAT 414..417 ACAT 348..349 AC 429..439 TCAACAC 546..547AC 348 ACAG (396) K326 18GH 414..417 ACAT 414..417 ACAT 348..349 AC429..439 TCAACAC 546..547 AC 349 ACAG (396) K326 18GH 414..417 ACAT414..417 ACAT 348..351 ACAC 431..438 AACACAC 546..547 AC 355 A 416..417AT 350..351 AC 435..438 CACA 550..553 ACAG 440..442 AGA K326 18GH414..417 ACAT 411..420 TCAACAT 348..349 AC 433..437 CACAC 548..552 ACACA359 ACA (379) 417..420 TACA K326 18GH 413..420 AACATACA 414..417 ACAT349..352 CACA 432..433 AC 546..549 ACAC 64 417..420 TACA 354..359 AGAGAAK326 18GH 415..421 CATACAG 413..422 AACATAC 348..351 ACAC 432..439ACACACA 543..554 TCAACA 682 AGA (386) G CACAGA (404) 417..420 TACA350..351 AC 437..438 CA 549..552 CACA K326 18GH 414..417 ACAT 414..420ACATACA 348..351 ACAC 432..439 ACACACA 546..549 ACAC 692 G 416..420ATACA 350..351 AC 437..438 CA K326 18GH 414..417 ACAT 414..417 ACAT348..351 ACAC 430..436 CAACACA 546..549 ACAC 697 K326 18GH 414..417 ACAT414..417 ACAT 348..351 ACAC 430..436 CAACACA 546..549 ACAC 922 K326 18GH414..417 ACAT 414..417 ACAT 349..355 CACACA 431..438 AACACAC 546..547 AC957 G A 351..354 CACA K326 17GH — — — — 346..350 CAACA 432..433 AC — —1886 349..350 CA K326 17GH — — 418..419 AC 348..349 AC — — — — 1888 K32617GH — — 418..419 AC 348..349 AC — — — — 1889 K326 17GH 413..419 AACATAC414..417 ACAT 348..351 ACAC 432..435 ACAC — — 1892 414..419 ACATAC350..351 AC 434..435 AC 416..419 ATAC K326 17GH 416..421 ATACAG 416..420ATACA 348..349 AC 430..436 CAACACA 546..547 AC 1893 K326 17GH 414..417ACAT 417..420 TACA 348..355 ACACAC 432..435 ACAC 548..552 ACACA 1901 AG434..435 AC 550..552 ACA K326 17GH 414..417 ACAT 417..420 TACA 348..355ACACAC 432..435 ACAC 548..552 ACACA 1902 AG 434..435 AC 550..552 ACAK326 17GH 414..417 ACAT 413..421 AACATAC 352..354 ACA 432..435 ACAC546..547 AC 1808 AG 418..421 ACAG 434..435 AC K326 17GH 414..417 ACAT417..420 TACA 348..355 ACACAC 432..435 ACAC 548..552 ACACA 1810 AG434..435 AC 550..552 ACA K326 18GH3 — — 414..417 ACAT 348..349 AC432..435 ACAC — — K326 18GH4 — — 414..417 ACAT 348..349 AC 429..439TCAACAC 546..547 AC ACAG (396)

TABLE 4BMutant pmt alleles in TN90 produced by genome editing using GET2. PMT1bPMT1a PMT2 PMT3 PMT4 Deleted Deleted Deleted Deleted Deleted VARIETYLINE Position sequence Position sequence Position sequence Positionsequence Position sequence TN90 17GH 414..417 ACAT 414..417 ACAT348..349 AC 436..439 ACAG 546..547 AC 1696 TN90 17GH 414..417 ACAT414..417 ACAT 346..352 CAACACA 432..435 ACAC 546..547 AC 1717 415..416CA 349..352 CACA 434..435 AC TN90 17GH 414..417 ACAT 417..420 TACA348..349 AC 432..433 AC 546..549 ACAC 1719 417..423 TACAGA G TN90 17GH412..421 CAACATA 412..418 CAACAT 348..351 ACAC 432..433 AC 546..547 AC1729 CAG (380) A 414..420 ACATACA TN90 17GH 418..421 ACAG 414..417 ACAT347..357 AACACACA 432..433 AC 546..547 AC 1736 GAG (391) 351..354 CACATN90 17GH 414..417 ACAT 414..417 ACAT 346..352 CAACACA 432..435 ACAC546..547 AC 1737 415..416 CA 349..352 CACA 434..435 AC TN90 17GH414..417 ACAT 414..417 ACAT 348..349 AC 432..433 AC 546..549 ACAC 1739548..549 AC TN90 17GH 414..417 ACAT 416..419 ATAC 348..351 ACAC 435..438CACA 546..547 AC 1740 418..419 AC 436..439 ACAG TN90 17GH 413..421AACATAC 417..420 TACA 350..354 ACACA 432..435 ACAC 546..547 AC 1835 AG417..420 TACA 418..421 ACAG 351..362 CACAGAGA ATGG (394) TN90 17GH414..417 ACAT 417..420 TACA 348..349 AC 432..433 AC 546..549 ACAC 1848417..423 TACAGA G TN90 17GH 414..417 ACAT 414..417 ACAT 348..351 ACAC430..436 CAACACA 546..549 ACAC 1849 433..436 CACA 548..549 AC TN90 17GH414..417 ACAT 417..420 TACA 348..349 AC 432..433 AC 546..549 ACAC 1912417..423 TACAGA G TN90 17GH 416..419 ATAC 412..421 CAACAT 348..351 ACAC432..435 ACAC 546..547 AC 1937 ACAG (380) 417..420 TACA 434..435 AC TN9017GH 414..417 ACAT 417..420 TACA 348..349 AC 432..433 AC 546..549 ACAC1940 417..423 TACAGA G TN90 17GH 414..417 ACAT 414..417 ACAT 348..349 AC432..433 AC 546..549 ACAC 1943 548..549 AC TN90 17GH 414..417 ACAT414..417 ACAT 348..349 AC 432..433 AC 546..549 ACAC 1944 548..549 ACTN90 18GH 414..418 ACATA 412..418 CAACAT 348..351 ACAC 432..433 AC546..549 ACAC 1051 A 415..421 CATACAG 415..418 CATA 350..351 AC 548..549AC TN90 18GH 416..419 ATAC 412..421 CAACAT 348..351 ACAC 432..435 ACAC546..547 AC 22 ACAG (380) 417..420 TACA 434..435 AC TN90 18GH 414..417ACAT 414..417 ACAT 348..349 AC 432..433 AC 546..549 ACAC 34 548..549 ACTN90 18GH 414..417 ACAT 414..417 ACAT 348..351 ACAC 430..436 CAACACA546..549 ACAC 473 433..436 CACA 548..549 AC TN90 18GH 412..421 CAACATA412..418 CAACAT 348..351 ACAC 432..433 AC 546..547 AC 49 CAG (380) ATN90 18GH 412..421 CAACATA 412..418 CAACAT 348..351 ACAC 432..433 AC546..547 AC 50 CAG (380) A 414..420 ACATACA TN90 18GH 414..418 ACATA412..418 CAACAT 348..351 ACAC 432..433 AC 546..549 ACAC 848 A 415..421CATACAG 415..418 CATA 350..351 AC 548..549 AC TN90 18GH 414..417 ACAT416..422 ATACAG 348..349 AC 435..438 CACA 546..547 AC 850 A 417..420TACA 436..439 ACAG 550..553 ACAG TN90 18GH 414..417 ACAT 416..422 ATACAG348..349 AC 435..438 CACA 546..547 AC 851 A 417..420 TACA 436..439 ACAG550..553 ACAG TN90 17GH 419..420 CA 413..420 AACATA 348..351 ACAC429..438 TCAACACAC 546..547 AC 1699 CA A (395) 418..423 ACAGAG 419..420CA 349..349 C 432..446 ACACACAG 427..427 G AGAATGG (399) TN90 17GH414..417 ACAT 414..415 AC 346..355 CAACACAC 432..437 ACACAC — — 1708418..424 ACAGAG AG (390) 440..443 AGAA A 419..420 CA TN90 17GH 415..420CATACA 414..417 ACAT 348..351 ACAC 432..433 AC 546..549 ACAC 1722418..421 ACAG 350..351 AC 435..439 CACAG TN90 17GH 416..421 ATACAG414..417 ACAT 348..351 ACAC 432..435 ACAC 550..553 ACAG 1724 417..420TACA 434..435 AC TN90 17GH 416..421 ATACAG 414..417 ACAT 348..351 ACAC432..435 ACAC 550..553 ACAG 1725 417..420 TACA 434..435 AC TN90 17GH416..418 ATA 412..418 CAACAT 348..351 ACAC 433..437 CACAC 546..551ACACAC 1845 A 418..419 AC 415..418 CATA 436..437 AC 550..551 AC TN9017GH 415..420 CATACA 414..417 ACAT 348..351 ACAC 432..433 AC 546..549ACAC 1846 418..421 ACAG 350..351 AC 435..439 CACAG TN90 17GH 415..420CATACA 414..417 ACAT 348..351 ACAC 432..433 AC 546..549 ACAC 1847418..421 ACAG 350..351 AC 435..439 CACAG TN90 17GH 414..417 ACAT417..420 TACA 348..349 AC 432..433 AC 546..549 ACAC 1911 417..423 TACAGAG TN90 17GH 414..417 ACAT 417..420 TACA 348..349 AC 432..433 AC 546..549ACAC 1912 417..423 TACAGA G TN90 17GH — — 414..417 ACAT — — 432..435ACAC — — 1915 TN90 17GH 414..419 ACATAC 417..420 TACA 353..361 CAGAGAAT432..435 ACAC 544..550 CAACAC 1918 G A 554..554 A 416..419 ATAC 418..421ACAG 558..563 TGGTGG 565..566 TT 569..572 CATA TN90 17GH 412..418CAACATA 414..417 ACAT — — 432..448 ACACACAG 546..547 AC 1928 AGAATGGTG (400) 415..418 CATA 419..421 CAG 437..438 CA TN90 17GH 414..419 ACATAC416..419 ATAC — — — — 546..551 ACACAC 1932 416..419 ATAC 418..419 ACTN90 17GH 414..419 ACATAC 416..419 ATAC 350..355 ACACAG 413..414 CT546..551 ACACAC 1933 418..419 GA 416..419 ATAC 418..419 AC 351..354 CACA426..427 AA 431..432 AA 432..435 ACAC TN90 17GH 413..421 AACATAC416..419 ATAC 348..351 ACAC 432..433 AC 544..550 CAACAC 1936 AG A417..420 TACA 418..419 AC 350..351 AC 547..550 CACA TN90 18GH 414..419ACATAC 348..349 AC 432..433 AC 546..549 ACAC 20 352..355 ACAG 548..549AC TN90 18GH 414..419 ACATAC 416..419 ATAC 348..349 AC 436..439 ACAG546..549 ACAC 47 418..419 AC 424..425 AA TN90 18GH 414..419 ACATAC414..417 ACAT 348..351 ACAC 433..437 CACAC 546..549 ACAC 28 416..419ATAC 350..351 AC 548..549 AC TN90 18GH 414..419 ACATAC 416..419 ATAC348..349 AC 436..439 ACAG 546..549 ACAC 31 418..419 AC 424..425 AA

TABLE 4CMutant pmt alleles in NLMz produced by genome editing using GET2. NLMz refers to the Narrow LeafMadole variety containing triple loss-of-function mutations in three nicotine demethylase genes(CYP82E4, CYP82E5v2, and CYP82E10). PMT1b PMT1a PMT2 PMT3 PMT4 DeletedDeleted Deleted Deleted Deleted VARIETY LINE Position sequence Positionsequence Position sequence Position sequence Position sequence NLMz 18GH414..417 ACAT 414..417 ACAT 350..351 AC 431..441 AACACACA 546..549 ACAC10 GAG (391) NLMz 18GH 414..417 ACAT 412..418 CAACATA 348..349 AC430..436 CAACACA 546..553 ACACACAG 1004 416..416 A 435..436 CA 551..552CA NLMz 18GH 414..417 ACAT 412..418 CAACATA 348..349 AC 430..436 CAACACA546..553 ACACACAG 1033 416..416 A 435..436 CA 551..552 CA NLMz 18GH417..418 TA 416..419 ATAC 348..352 ACACA 432..437 ACACAC 546..547 AC 132418..419 AC 348..353 ACACAC 434..437 ACAC 436..437 AC NLMz 18GH 414..417ACAT 414..423 ACATACAG 348..351 ACAC 433..439 CACACAG 550..556 ACAGAGA134 AG (388) 419..420 CA NLMz 18GH 414..417 ACAT 415..419 CATAC 346..352CAACACA 432..435 ACAC 545..557 AACACACA 217 GAGAA (407) 417..418 TA351..352 CA 551..552 CA NLMz 18GH 416..419 ATAC 414..417 ACAT 348..349AC 432..433 AC 546..549 ACAC 456 418..419 AC NLMz 18GH 414..417 ACAT414..417 ACAT 348..349 AC 432..433 AC 546..549 ACAC 457 NLMz 18GH414..417 ACAT 409..420 ATTCAACA 350..363 ACACAGA 436..439 ACAG 550..553ACAG 460 TACA (383) GAATGGT (393) 416..429 ATACAGAG 351..354 CACA AATGGT353..354 CA (389) NLMz 18GH 414..417 ACAT 412..418 CAACATA 348..349 AC430..436 CAACACA 546..553 ACACACAG 465 416..416 A 435..436 CA 551..552CA NLMz 18GH 414..417 ACAT 414..417 ACAT 348..351 ACAC 432..435 ACAC550..553 ACAG 830 NLMz 18GH 413..428 AACATACAGA 413..428 AACATACA348..351 ACAC 546..547 AC 831 GAATGG (381) GAGAATGG (381) 417..420 TACANLMz 18GH 414..417 ACAT 414..417 ACAT 348..349 AC 432..433 AC 546..549ACAC 836 NLMz 18GH 415..421 CATACAG 413..420 AACATACA 347..357 AACACAC546..547 AC 841 419..420 CA 414..420 ACATACA AGAG (391) NLMz 18GH411..420 TCAACATACA 411..420 TCAACATA 351..354 CACA 432..433 AC 546..549ACAC 974 (379) CA (379) 417..420 TACA 353..354 CA 436..439 ACAG NLMz18GH 412..418 CAACATA 414..417 ACAT 346..352 CAACACA 429..439 TCAACACA546..552 ACACACA 981 CAG (396) 349..352 CACA 431..441 AACACACA 546..553ACACACAG GAG (391) NLMz 18GH 414..417 ACAT 412..418 CAACATA 348..349 AC430..436 CAACACA 546..553 ACACACAG 994 416..416 A 435..436 CA 551..552CA NLMz 18GH — — 414..420 ACATACA 348..351 ACAC — — 546..547 AC 128417..421 TACAG 350..351 AC NLMz 18GH 414..437 ACATACAGAG 412..418CAACATA 349..355 CACACAG 430..436 CAACACA 546..549 ACAC 130 AATGGTGGATTTCC (382) 417..420 TACA 415..418 CATA NLMz 18GH 417..418 TA 416..419ATAC 348..353 ACACAC — — 546..547 AC 131 417..419 TAC 350..353 ACAC418..419 AC NLMz 18GH 413..419 AACATAC 414..420 ACATACA 348..351 ACAC —— 546..547 AC 133 414..419 ACATAC 417..420 TACA 350..351 AC 417..421TACAG NLMz 18GH — — — — 348..349 AC — — — — 136 NLMz 18GH 412..418CAACATA 414..419 ACATAC 347..354 AACACAC 432..433 AC 546..549 ACAC 216415..418 CATA 416..419 ATAC A 548..549 AC NLMz 18GH 418..419 AC 414..417ACAT 348..351 ACAC 546..549 ACAC 227 NLMz 18GH 414..417 ACAT 414..420ACATACA 352..355 ACAG 429..435 TCAACAC 546..551 ACACAC 5 415..421CATACAG 432..435 ACAC 548..551 ACAC NLMz 18GH 414..417 ACAT 416..429ATACAGAG 350..363 ACACAGA 436..439 ACAG 550..553 ACAG 6 AATGGT GAATGGT(389) (393) 417..422 TACAGA 351..356 CACAGA 353..356 CAGA NLMz 18GH416.423 ATACAGAG 414..419 ACATAC 348..351 ACAC 433..437 CACAC 546..549ACAC 65 418.420 ACA 417..419 TAC 350..351 AC 436..437 AC NLMz 18GH414.417 ACAT 414..420 ACATACA 352..355 ACAG 429..435 TCAACAC 546..551ACACAC 66 415.421 CATACAG 432..435 ACAC 548..551 ACAC NLMz 18GH 411..420TCAACATA 432..433 AC 546..549 ACAC 69 CA (379) 436..439 ACAG NLMz 18GH416..423 ATACAGAG 414..419 ACATAC 348..351 ACAC 433..437 CACAC 546..549ACAC 72 418..420 ACA 417..419 TAC 350..351 AC 436..437 AC NLMz 18GH — —414..420 ACATACA 348..351 ACAC — — 546..547 AC 73 417..421 TACAG350..351 AC NLMz 18GH — — 412..418 CAACATA — — — — — — 74 NLMz 18GH414..419 ACATAC 414..417 ACAT 348..349 AC 431..431 A 546..549 ACAC 78434..438 ACACA 548..549 AC 435..438 CACA NLMz 18GH — — 414..420 ACATACA348..351 ACAC — — 546..547 AC 79 417..421 TACAG 350..351 AC NLMz 18GH417..420 TACA 416..421 ATACAG 348..354 ACACACA 435..447 CACAGAGA549..552 CACA 8 417..420 TACA 350..354 ACACA ATGGT 549..553 CACAG (401)NLMz 18GH 417..418 TA 416..419 ATAC 348..353 ACACAC — — 546..547 AC 9418..419 AC NLMz 18GH 414..417 ACAT 414..417 ACAT 348..351 ACAC 431..441AACACACA 546..549 ACAC 71 GAG (391) NLMz 17GH 414..417 ACAT 414..420ACATACA 352..355 ACAG 429..435 TCAACAC 546..551 ACACAC 1905 415..421CATACAG 432..435 ACAC 548..551 ACAC

TABLE 4DMutant pmt alleles in oriental tobacco produced by genome editing using GET2.PMT1b PMT1a PMT2 PMT3 PMT4 Deleted Deleted Deleted Deleted DeletedVARIETY LINE Position sequence Position sequence Position sequencePosition sequence Position sequence Katerini 18GH125 412..418 CAACATA416..419 ATAC 348..355 ACACACAG 432..435 ACAC 546..547 AC 414..418 ACATA418..419 AC 434..435 AC Basma 18GH203 414..417 ACAT 412..418 CAACATA348..357 ACACACAG 432..435 ACAC 546..549 ACAC AG (392) 415..418 CATA353..354 CA 434..435 AC Katerini 18GH208 416..419 ATAC 414..417 ACAT352..355 ACAG 432..441 ACACACAG 546..553 ACACAC AG (392) AG 418..419 AC435..438 CACA Basma 18GH341 414..417 ACAT 412..418 CAACATA 348..357ACACACAG 432..435 ACAC 546..549 ACAC AG (392) 415..418 CATA 353..354 CA434..435 AC Katerini 18GH403 414..417 ACAT 414..417 ACAT 348..351 ACAC432..435 ACAC Katerini 18GH414 412..418 CAACATA 414..417 ACAT 348..349AC 430..436 CAACACA 546..547 AC 415..418 CATA Katerini 18GH434 413..420AACATAC 414..417 ACAT 351..357 CACAGAG 433..439 CACACAG 546..547 AC A417..420 TACA 437..438 CA Katerini 18GH436 412..418 CAACATA 414..421ACATACA 346..352 CAACACA 432..433 AC 546..547 AC G 436..443 ACAGAGAA415..416 CA 445..449 GGTGG 451..463 TTTCCATAC ACTG (402) Katerini18GH437 414..417 ACAT 414..417 ACAT 348..349 AC 433..439 CACACAG544..553 CAACAC ACAG (390) 416..417 AT 435..438 CACA 551..552 CAKaterini 18GH449 414..417 ACAT 413..419 AACATAC 348..349 AC 432..435ACAC 546..549 ACAC 415..416 CA 418..419 AC Katerini 18GH706 416..420ATACA 414..417 ACAT 348..351 ACAC 432..439 ACACACAG 545..555 AACACA419..420 CA 435..438 CACA CAGAG (391) Katerini 18GH709 412..418 CAACATA414..417 ACAT 348..349 AC 432..435 ACAC 546..547 AC 417..418 TA 416..417AT Katerini 18GH710 414..417 ACAT 414..417 ACAT 351..354 CACA 432..435ACAC 546..547 AC 352..355 ACAG 434..435 AC Katerini 18GH716 416..419ATAC 417..420 TACA 348..351 ACAC 432..439 ACACACAG 546..549 ACAC418..419 AC 417..421 TACAG 435..438 CACA 548..549 AC Katerini 18GH729414..417 ACAT 414..417 ACAT 350..357 ACACAGAG 432..435 ACAC 546..547 AC353..354 CA Katerini 18GH731 414..417 ACAT 414..417 ACAT 348..351 ACAC433..433 C 544..553 CAACAC 435..439 CACAG ACAG (390) Katerini 18GH752416..419 ATAC 414..417 ACAT 348..349 AC 432..435 ACAC 546..549 ACAC418..419 AC Katerini 18GH756 416..419 ATAC 414..417 ACAT 353..354 CA433..439 CACACAG 545..555 AACACA CAGAG (391) 416..419 ATAC 414..417 ACAT353..354 CA 433..439 CACACAG 545..555 AACACA CAGAG (391) 437..438 CA549..552 CACA Katerini 18GH768 416..419 ATAC 414..417 ACAT 348..351 ACAC432..433 AC 544..550 CAACAC A 547..550 CACA Katerini 18GH771 416..419ATAC 414..417 ACAT 346..352 CAACACA 432..435 ACAC 546..547 AC 349..352CACA 434..435 AC Katerini 18GH776 409..415 ATTCAAC 411..417 TCAACAT348..351 ACAC 441..441 G 546..549 ACAC 418..419 AC 414..417 ACAT548..549 AC Katerini 18GH800 412..418 CAACATA 414..420 ACATACA 348..351ACAC 432..435 ACAC 541..551 ATTCAAC 415..418 CATA 416..420 ATACA ACAC(403) 548..551 ACAC Katerini 18GH818 414..417 ACAT 409..415 ATTCAAC348..351 ACAC 432..435 ACAC — — 434..435 AC

TABLE 4E Mutant pmt alleles in NLM (Ph Ph) tobacco produced by genomeediting using GET1. PMT1b PMT1a PMT2 PMT3 PMT4 Position PositionPosition Position from Position LINE Modification from ATG from ATG fromATG ATG from ATG CS15 A-deleted 131 A- 132 A-deleted 98 A-deleted 98A-del 131 T-deleted 262 inserted T-deleted 196 T-deleted 282

TABLE 5AA list of exemplary mutant alleles obtained in the PMT1b gene. Mutant allele sequences listed hereand Tables 5B to 5E represent about 40-nucleotide-long genomic sequences from each edited PMT genewith the edited site in the middle of the genomic sequence (e.g., 20 nucleotides on each side ofthe deleted or inserted sequence site). These mutant alleles corresponds to those listed in Tables4A to 4E. PMT1b Mutant Reference Allele Allele Deleted sequence sequencesequence (SEQ ID Mutant (SEQ ID Position (SEQ ID No.) No.)Allele Sequence No.) Reference Allele Sequence 131...131 A 23TGGCATTTCCAAACACCAA 201 TGGCATTTCCAAACACCAAAaCGGGCACCAACGGGCACCAGAATGGCAC GAATGGCACTT TT 262...262 T 24 CCAACTCTATTAAGCCTGGT202 CCAACTCTATTAAGCCTGGTtGGTTTTCAGA GGTTTTCAGAGTTTAGCGCA GTTTAGCGCA409..415 ATTCAAC 25 TTCTGACTTTGGATGGAGCA 203TTCTGACTTTGGATGGAGCAattcaacATACAG ATACAGAGAATGGTGGATT AGAATGGTGGATTT T411..420 TCAACATACA 26 CTGACTTTGGATGGAGCAA 204CTGACTTTGGATGGAGCAATtcaacatacaGAGA (379) TGAGAATGGTGGATTTCCAATGGTGGATTTCCATA TA 412..418 CAACATA 27 TGACTTTGGATGGAGCAAT 205TGACTTTGGATGGAGCAATTcaacataCAGAGA TCAGAGAATGGTGGATTTC ATGGTGGATTTCCA CA412..421 CAACATACAG 28 TGACTTTGGATGGAGCAAT 206TGACTTTGGATGGAGCAATTcaacatacagAGAA (380) TAGAATGGTGGATTTCCATTGGTGGATTTCCATAC AC 413..419 AACATAC 29 GACTTTGGATGGAGCAATT 207GACTTTGGATGGAGCAATTCaacatacAGAGAA CAGAGAATGGTGGATTTCC TGGTGGATTTCCAT AT413..420 AACATACA 30 GACTTTGGATGGAGCAATT 208GACTTTGGATGGAGCAATTCaacatacaGAGAA CGAGAATGGTGGATTTCCA TGGTGGATTTCCATA TA413..421 AACATACAG 31 GACTTTGGATGGAGCAATT 209GACTTTGGATGGAGCAATTCaacatacagAGAA CAGAATGGTGGATTTCCAT TGGTGGATTTCCATACAC 413..428 AACATACAGAGA 32 GACTTTGGATGGAGCAATT 210GACTTTGGATGGAGCAATTCaacatacagagaatgg ATGG (381) CTGGATTTCCATACACTGAATGGATTTCCATACACTGAAA A 414..417 ACAT 33 ACTTTGGATGGAGCAATTC 211ACTTTGGATGGAGCAATTCAacatACAGAGA AtACAGAGAATGGTGGATTT ATGGTGGATTTCC CC414..418 ACATA 34 ACTTTGGATGGAGCAATTC 212ACTTTGGATGGAGCAATTCAacataCAGAGAA ACAGAGAATGGTGGATTTC TGGTGGATTTCCA CA414..419 ACATAC 35 ACTTTGGATGGAGCAATTC 213ACTTTGGATGGAGCAATTCAacatacAGAGAA AAGAGAATGGTGGATTTCC TGGTGGATTTCCAT AT414..420 ACATACA 36 ACTTTGGATGGAGCAATTC 214ACTTTGGATGGAGCAATTCAacatacaGAGAAT AGAGAATGGTGGATTTCCA GGTGGATTTCCATA TA414..437 ACATACAGAGAA 37 ACTTTGGATGGAGCAATTC 215ACTTTGGATGGAGCAATTCAacatacagagaatggt TGGTGGATTTCC AATACACTGAAATGATTGTggatttccATACACTGAAATGATTGTTC (382) TC 415..416 CA 38 CTTTGGATGGAGCAATTCA216 CTTTGGATGGAGCAATTCAAcaTACAGAGAA ATACAGAGAATGGTGGATT TGGTGGATTTC TC415..418 CATA 39 CTTTGGATGGAGCAATTCA 217CTTTGGATGGAGCAATTCAAcataCAGAGAAT ACAGAGAATGGTGGATTTC GGTGGATTTCCA CA415..420 CATACA 40 CTTTGGATGGAGCAATTCA 218CTTTGGATGGAGCAATTCAAcatacaGAGAAT AGAGAATGGTGGATTTCCA GGTGGATTTCCATA TA415..421 CATACAG 41 CTTTGGATGGAGCAATTCA 219CTTTGGATGGAGCAATTCAAcatacagAGAATG AAGAATGGTGGATTTCCAT GTGGATTTCCATAC AC416..416 A 42 TTTGGATGGAGCAATTCAA 220 TTTGGATGGAGCAATTCAACaTACAGAGAACTACAGAGAATGGTGGATT TGGTGGATTTC TC 416..418 ATA 43 TTTGGATGGAGCAATTCAA221 TTTGGATGGAGCAATTCAACataCAGAGAAT CCAGAGAATGGTGGATTTC GGTGGATTTCCA CA416..419 ATAC 44 TTTGGATGGAGCAATTCAA 222TTTGGATGGAGCAATTCAACatacAGAGAATG CAGAGAATGGTGGATTTCC GTGGATTTCCAT AT416..420 ATACA 45 TTTGGATGGAGCAATTCAA 223TTTGGATGGAGCAATTCAACatacaGAGAATG CGAGAATGGTGGATTTCCA GTGGATTTCCATA TA416..421 ATACAG 46 TTTGGATGGAGCAATTCAA 224TTTGGATGGAGCAATTCAACatacagAGAATG CAGAATGGTGGATTTCCAT GTGGATTTCCATAC AC416..423 ATACAGAG 47 TTTGGATGGAGCAATTCAA 225TTTGGATGGAGCAATTCAACatacagagAATGG CAATGGTGGATTTCCATAC TGGATTTCCATACAC AC417..418 TA 48 TTGGATGGAGCAATTCAAC 226 TTGGATGGAGCAATTCAACAtaCAGAGAATGACAGAGAATGGTGGATTTC GTGGATTTCCA CA 417..420 TACA 49 TTGGATGGAGCAATTCAAC227 TTGGATGGAGCAATTCAACAtacaGAGAATG AGAGAATGGTGGATTTCCA GTGGATTTCCATA TA418..419 AC 50 TGGATGGAGCAATTCAACA 228 TGGATGGAGCAATTCAACATacAGAGAATGTAGAGAATGGTGGATTTCC GTGGATTTCCAT AT 418..420 ACA 51 TGGATGGAGCAATTCAACA229 TGGATGGAGCAATTCAACATacaGAGAATGG TGAGAATGGTGGATTTCCA TGGATTTCCATA TA418..421 ACAG 52 TGGATGGAGCAATTCAACA 230 TGGATGGAGCAATTCAACATacagAGAATGGTAGAATGGTGGATTTCCAT TGGATTTCCATAC AC 418..423 ACAGAG 53TGGATGGAGCAATTCAACA 231 TGGATGGAGCAATTCAACATacagagAATGGTTAATGGTGGATTTCCATACA GGATTTCCATACAC C 419..420 CA 54 GGATGGAGCAATTCAACAT232 GGATGGAGCAATTCAACATAcaGAGAATGG AGAGAATGGTGGATTTCCA TGGATTTCCATA TA427..427 G 55 CAATTCAACATACAGAGAA 233 CAATTCAACATACAGAGAATgGTGGATTTCTGTGGATTTCCATACACTGA CATACACTGAA A

TABLE 5B A list of exemplary mutant alleles obtained in the PMT1a gene.PMT1a Mutant Reference Allele Allele Deleted sequence sequence sequence(SEQ ID Mutant (SEQ ID Position (SEQ ID No.) No.) Allele Sequence No.)Reference Allele Sequence 132...132 A inserted 56 GCACTTCCAAACACCAAAACa234 GCACTTCCAAACACCAAAACGGGCA GGGCACCAGAATGGCACTTT CCAGAATGGCACTTT409..415 ATTCAAC 57 TTCTGACTTTGGATGGAGCAA 235TTCTGACTTTGGATGGAGCAattcaacAT TACAGAGAATGGTGGATTT ACAGAGAATGGTGGATTT409..420 ATTCAACATACA 58 TTCTGACTTTGGATGGAGCAG 236TTCTGACTTTGGATGGAGCAattcaacatac (383) AGAATGGTGGATTTCCATAaGAGAATGGTGGATTTCCATA 411..417 TCAACAT 59 CTGACTTTGGATGGAGCAATA 237CTGACTTTGGATGGAGCAATtcaacatAC CAGAGAATGGTGGATTTCC AGAGAATGGTGGATTTCC411..420 TCAACATACA 60 CTGACTTTGGATGGAGCAATG 238CTGACTTTGGATGGAGCAATtcaacataca (384) AGAATGGTGGATTTCCATAGAGAATGGTGGATTTCCATA 412..418 CAACATA 61 TGACTTTGGATGGAGCAATTC 239TGACTTTGGATGGAGCAATTcaacataCA AGAGAATGGTGGATTTCCA GAGAATGGTGGATTTCCA412..421 CAACATACAG 62 TGACTTTGGATGGAGCAATTA 240TGACTTTGGATGGAGCAATTcaacatacag (385) GAATGGTGGATTTCCATACAGAATGGTGGATTTCCATAC 413..419 AACATAC 63 GACTTTGGATGGAGCAATTCA 241GACTTTGGATGGAGCAATTCaacatacA GAGAATGGTGGATTTCCAT GAGAATGGTGGATTTCCAT413..420 AACATACA 64 GACTTTGGATGGAGCAATTCG 242GACTTTGGATGGAGCAATTCaacatacaG AGAATGGTGGATTTCCATA AGAATGGTGGATTTCCATA413..421 AACATACAG 65 GACTTTGGATGGAGCAATTCA 243GACTTTGGATGGAGCAATTCaacatacag GAATGGTGGATTTCCATAC AGAATGGTGGATTTCCATAC413..422 AACATACAGA 66 GACTTTGGATGGAGCAATTCG 244GACTTTGGATGGAGCAATTCaacatacag (386) AATGGTGGATTTCCATACAaGAATGGTGGATTTCCATACA 413..428 AACATACAGAG 67 GACTTTGGATGGAGCAATTCT 245GACTTTGGATGGAGCAATTCaacatacag AATGG (387) GGATTTCCATACACTGAAAagaatggTGGATTTCCATACACTGAAA 414..415 AC 68 ACTTTGGATGGAGCAATTCAA 246ACTTTGGATGGAGCAATTCAacATAC TACAGAGAATGGTGGATTT AGAGAATGGTGGATTT 414..417ACAT 69 ACTTTGGATGGAGCAATTCAA 247 ACTTTGGATGGAGCAATTCAacatACACAGAGAATGGTGGATTTCC GAGAATGGTGGATTTCC 414..419 ACATAC 70ACTTTGGATGGAGCAATTCAA 248 ACTTTGGATGGAGCAATTCAacatacAGGAGAATGGTGGATTTCCAT AGAATGGTGGATTTCCAT 414..420 ACATACA 71ACTTTGGATGGAGCAATTCAG 249 ACTTTGGATGGAGCAATTCAacatacaGAGAATGGTGGATTTCCATA AGAATGGTGGATTTCCATA 414..421 ACATACAG 72ACTTTGGATGGAGCAATTCAA 250 ACTTTGGATGGAGCAATTCAacatacagAGAATGGTGGATTTCCATAC GAATGGTGGATTTCCATAC 414..423 ACATACAGAG 73ACTTTGGATGGAGCAATTCAA 251 ACTTTGGATGGAGCAATTCAacatacaga (388)ATGGTGGATTTCCATACAC gAATGGTGGATTTCCATACAC 415..415 C 74CTTTGGATGGAGCAATTCAAA 252 CTTTGGATGGAGCAATTCAAcATACA TACAGAGAATGGTGGATTTGAGAATGGTGGATTT 415..416 CA 75 CTTTGGATGGAGCAATTCAAT 253CTTTGGATGGAGCAATTCAAcaTACA ACAGAGAATGGTGGATTTC GAGAATGGTGGATTTC 415..418CATA 76 CTTTGGATGGAGCAATTCAAC 254 CTTTGGATGGAGCAATTCAAcataCAGAGAGAATGGTGGATTTCCA AGAATGGTGGATTTCCA 415..419 CATAC 77CTTTGGATGGAGCAATTCAAA 255 CTTTGGATGGAGCAATTCAAcatacAGAGAGAATGGTGGATTTCCAT GAATGGTGGATTTCCAT 415..421 CATACAG 78CTTTGGATGGAGCAATTCAAA 256 CTTTGGATGGAGCAATTCAAcatacagAGAATGGTGGATTTCCATAC GAATGGTGGATTTCCATAC 416..417 AT 79TTTGGATGGAGCAATTCAACtA 257 TTTGGATGGAGCAATTCAACatACAGACAGAGAATGGTGGATTTCC GAATGGTGGATTTCC 416..419 ATAC 80TTTGGATGGAGCAATTCAACA 258 TTTGGATGGAGCAATTCAACatacAGAGAGAATGGTGGATTTCCAT GAATGGTGGATTTCCAT 416..420 ATACA 81TTTGGATGGAGCAATTCAACG 259 TTTGGATGGAGCAATTCAACatacaGAGAGAATGGTGGATTTCCATA AATGGTGGATTTCCATA 416..421 ATACAG 82TTTGGATGGAGCAATTCAACA 260 TTTGGATGGAGCAATTCAACatacagAGGAATGGTGGATTTCCATAC AATGGTGGATTTCCATAC 416..422 ATACAGA 83TTTGGATGGAGCAATTCAACG 261 TTTGGATGGAGCAATTCAACatacagaGAATGGTGGATTTCCATACA AATGGTGGATTTCCATACA 416..429 ATACAGAGAAT 84TTTGGATGGAGCAATTCAACG 262 TTTGGATGGAGCAATTCAACatacagaga GGT (389)GATTTCCATACACTGAAAT atggtGGATTTCCATACACTGAAAT 417..417 T 85TTGGATGGAGCAATTCAACAA 263 TTGGATGGAGCAATTCAACAtACAGA CAGAGAATGGTGGATTTCCGAATGGTGGATTTCC 417..418 TA 86 TTGGATGGAGCAATTCAACAC 264TTGGATGGAGCAATTCAACAtaCAGA AGAGAATGGTGGATTTCCA GAATGGTGGATTTCCA 417..419TAC 87 TTGGATGGAGCAATTCAACAA 265 TTGGATGGAGCAATTCAACAtacAGAGGAGAATGGTGGATTTCCAT AATGGTGGATTTCCAT 417..420 TACA 88TTGGATGGAGCAATTCAACAG 266 TTGGATGGAGCAATTCAACAtacaGAGAGAATGGTGGATTTCCATA AATGGTGGATTTCCATA 417..421 TACAG 89TTGGATGGAGCAATTCAACAA 267 TTGGATGGAGCAATTCAACAtacagAGGAATGGTGGATTTCCATAC AATGGTGGATTTCCATAC 417..422 TACAGA 90TTGGATGGAGCAATTCAACAG 268 TTGGATGGAGCAATTCAACAtacagaGAAATGGTGGATTTCCATACA ATGGTGGATTTCCATACA 417.423 TACAGAG 91TTGGATGGAGCAATTCAACAA 269 TTGGATGGAGCAATTCAACAtacagagAATGGTGGATTTCCATACAC ATGGTGGATTTCCATACAC 418..419 AC 92TGGATGGAGCAATTCAACATA 270 TGGATGGAGCAATTCAACATacAGAG GAGAATGGTGGATTTCCATAATGGTGGATTTCCAT 418..421 ACAG 93 TGGATGGAGCAATTCAACATA 271TGGATGGAGCAATTCAACATacagAGA GAATGGTGGATTTCCATAC ATGGTGGATTTCCATAC418..424 ACAGAGA 94 TGGATGGAGCAATTCAACATA 272TGGATGGAGCAATTCAACATacagagaA TGGTGGATTTCCATACACT TGGTGGATTTCCATACACT419..420 CA 95 GGATGGAGCAATTCAACATAG 273 GGATGGAGCAATTCAACATAcaGAGAAGAATGGTGGATTTCCATA ATGGTGGATTTCCATA 419..421 CAG 96GGATGGAGCAATTCAACATAA 274 GGATGGAGCAATTCAACATAcagAGA GAATGGTGGATTTCCATACATGGTGGATTTCCATAC 424..425 AA 97 GAGCAATTCAACATACAGAGT 275GAGCAATTCAACATACAGAGaaTGGT GGTGGATTTCCATACACTG GGATTTCCATACACTG

TABLE 5C A list of exemplary mutant alleles obtained in the PMT2 gene.PMT2 Mutant Reference Allele Allele Deleted sequence sequence sequence(SEQ ID Mutant (SEQ ID Position (SEQ ID No.) No.) Allele Sequence No.)Reference Allele Sequence 98...98 A  98 TGGCACTTCCAAACACCA 276TGGCACTTCCAAACACCAAAaCG AACGGCCACAAGAATGGG GCCACAAGAATGGGACTT ACTT196...619 T  99 CCAATTGTATTAAGCCTGG 277 CCAATTGTATTAAGCCTGGTtGGTGGTTTTCAGAGTTTAGCG TTTTCAGAGTTTAGCGCA CA 346..350 CAACA 100TGACTTTGGATGGAGCAAT 278 TGACTTTGGATGGAGCAATTcaac TCACAGAGAATGGTGGATaCACAGAGAATGGTGGATTTC TTC 346..352 CAACACA 101 TGACTTTGGATGGAGCAAT 279TGACTTTGGATGGAGCAATTcaac TCAGAGAATGGTGGATTTC acaCAGAGAATGGTGGATTTCCA CA346..355 CAACACACA 102 TGACTTTGGATGGAGCAAT 280 TGACTTTGGATGGAGCAATTcaacG (390) TAGAATGGTGGATTTCCAT acacagAGAATGGTGGATTTCCATA AC C 347..354AACACACA 103 GACTTTGGATGGAGCAATT 281 GACTTTGGATGGAGCAATTCaacaCGAGAATGGTGGATTTCC cacaGAGAATGGTGGATTTCCATA ATA 347..357 AACACACAG 104GACTTTGGATGGAGCAATT 282 GACTTTGGATGGAGCAATTCaaca AG (391)CAATGGTGGATTTCCATAC cacagagAATGGTGGATTTCCATAC AC AC 348..349 AC 105ACTTTGGATGGAGCAATTC 283 ACTTTGGATGGAGCAATTCAacA AACACAGAGAATGGTGGACACAGAGAATGGTGGATTT TTT 348..351 ACAC 106 ACTTTGGATGGAGCAATTC 284ACTTTGGATGGAGCAATTCAacac AACAGAGAATGGTGGATT ACAGAGAATGGTGGATTTCC TCC348..352 ACACA 107 ACTTTGGATGGAGCAATTC 285 ACTTTGGATGGAGCAATTCAacacACAGAGAATGGTGGATTT aCAGAGAATGGTGGATTTCCA CCA 348..353 ACACAC 108ACTTTGGATGGAGCAATTC 286 ACTTTGGATGGAGCAATTCAacac AAGAGAATGGTGGATTTCacAGAGAATGGTGGATTTCCAT CAT 348..354 ACACACA 109 ACTTTGGATGGAGCAATTC 287ACTTTGGATGGAGCAATTCAacac AGAGAATGGTGGATTTCC acaGAGAATGGTGGATTTCCATA ATA348..355 ACACACAG 110 ACTTTGGATGGAGCAATTC 288 ACTTTGGATGGAGCAATTCAacacAAGAATGGTGGATTTCCAT acagAGAATGGTGGATTTCCATAC AC 348..357 ACACACAGA 111ACTTTGGATGGAGCAATTC 289 ACTTTGGATGGAGCAATTCAacac G (392)AAATGGTGGATTTCCATAC acagagAATGGTGGATTTCCATACA AC C 349..349 C 112CTTTGGATGGAGCAATTCA 290 CTTTGGATGGAGCAATTCAAcAC AACACAGAGAATGGTGGAACAGAGAATGGTGGATTT TTT 349..350 CA 113 CTTTGGATGGAGCAATTCA 291CTTTGGATGGAGCAATTCAAcaC ACACAGAGAATGGTGGAT ACAGAGAATGGTGGATTTC TTC349..352 CACA 114 CTTTGGATGGAGCAATTCA 292 CTTTGGATGGAGCAATTCAAcacaACAGAGAATGGTGGATTT CAGAGAATGGTGGATTTCCA CCA 349..355 CACACAG 115CTTTGGATGGAGCAATTCA 293 CTTTGGATGGAGCAATTCAAcaca AAGAATGGTGGATTTCCATcagAGAATGGTGGATTTCCATAC AC 350..351 AC 116 TTTGGATGGAGCAATTCAA 294TTTGGATGGAGCAATTCAACacA CACAGAGAATGGTGGATT CAGAGAATGGTGGATTTCC TCC350..353 ACAC 117 TTTGGATGGAGCAATTCAA 295 TTTGGATGGAGCAATTCAACacacCAGAGAATGGTGGATTTC AGAGAATGGTGGATTTCCAT CAT 350..354 ACACA 118TTTGGATGGAGCAATTCAA 296 TTTGGATGGAGCAATTCAACacac CGAGAATGGTGGATTTCCaGAGAATGGTGGATTTCCATA ATA 350..355 ACACAG 119 TTTGGATGGAGCAATTCAA 297TTTGGATGGAGCAATTCAACacac CAGAATGGTGGATTTCCAT agAGAATGGTGGATTTCCATAC AC350..357 ACACAGAG 120 TTTGGATGGAGCAATTCAA 298 TTTGGATGGAGCAATTCAACacacCAATGGTGGATTTCCATAC agagAATGGTGGATTTCCATACAC AC 350..363 ACACAGAGA 121TTTGGATGGAGCAATTCAA 299 TTTGGATGGAGCAATTCAACacac ATGGT (393)CGGATTTCCATACACTGAA agagaatggtGGATTTCCATACACTG AT AAAT 351..352 CA 122TTGGATGGAGCAATTCAA 300 TTGGATGGAGCAATTCAACAcaC CACAGAGAATGGTGGATTAGAGAATGGTGGATTTCCA TCCA 351..354 CACA 123 TTGGATGGAGCAATTCAA 301TTGGATGGAGCAATTCAACAcaca CAGAGAATGGTGGATTTC GAGAATGGTGGATTTCCATA CATA351..356 CACAGA 124 TTGGATGGAGCAATTCAA 302 TTGGATGGAGCAATTCAACAcacaCAGAATGGTGGATTTCCAT gaGAATGGTGGATTTCCATACA ACA 351..357 CACAGAG 125TTGGATGGAGCAATTCAA 303 TTGGATGGAGCAATTCAACAcaca CAAATGGTGGATTTCCATAgagAATGGTGGATTTCCATACAC CAC 351..362 CACAGAGAA 126 TTGGATGGAGCAATTCAA304 TTGGATGGAGCAATTCAACAcaca TGG (394) CATGGATTTCCATACACTGgagaatggTGGATTTCCATACACTGA AAA AA 352..354 ACA 127 TGGATGGAGCAATTCAAC305 TGGATGGAGCAATTCAACACaca ACGAGAATGGTGGATTTC GAGAATGGTGGATTTCCATA CATA352..355 ACAG 128 TGGATGGAGCAATTCAAC 306 TGGATGGAGCAATTCAACACacagACAGAATGGTGGATTTCC AGAATGGTGGATTTCCATAC ATAC 353..354 CA 129GGATGGAGCAATTCAACA 307 GGATGGAGCAATTCAACACAcaG CAGAGAATGGTGGATTTCAGAATGGTGGATTTCCATA CATA 353..356 CAGA 130 GGATGGAGCAATTCAACA 308GGATGGAGCAATTCAACACAcaga CAGAATGGTGGATTTCCAT GAATGGTGGATTTCCATACA ACA353..361 CAGAGAATG 131 GGATGGAGCAATTCAACA 309 GGATGGAGCAATTCAACACAcagaCAGTGGATTTCCATACACT gaatgGTGGATTTCCATACACTGAA GAA 354..359 AGAGAA 132GATGGAGCAATTCAACAC 310 GATGGAGCAATTCAACACACagag ACTGGTGGATTTCCATACAaaTGGTGGATTTCCATACACTG CTG

TABLE 5D A list of exemplary mutant alleles obtained in the PMT3 gene.PT3 Mutant Reference Allele Allele Deleted sequence sequence sequence(SEQ ID Mutant (SEQ ID Position (SEQ ID No.) No.) Allele Sequence No.)Reference Allele Sequence 98...98 A 133 TGGCACTTCCAAACACCAAACGGC 311TGGCACTTCCAAACACCAAAaCGGCCA CACCAGAATGGCACTT CCAGAATGGCACTT 280...280 T134 CCAACTCTATTAAGCCTGGTGGTTT 312 CCAACTCTATTAAGCCTGGTtGGTTTTCTCAGAGTTTAGCGCA AGAGTTTAGCGCA 413..414 CT 135 AACATATGGGAAGGTTCTGATTGG313 AACATATGGGAAGGTTCTGActTTGGAT ATGGAGCAATTCAACA GGAGCAATTCAACA418..419 GA 136 ATGGGAAGGTTCTGACTTTGTGGA 314ATGGGAAGGTTCTGACTTTGgaTGGAGC GCAATTCAACACACAG AATTCAACACACAG 426..427 AA137 GTTCTGACTTTGGATGGAGCTTCA 315 GTTCTGACTTTGGATGGAGCaaTTCAACACACACAGAGAATGGT ACACAGAGAATGGT 429..435 TCAACAC 138CTGACTTTGGATGGAGCAATACAG 316 CTGACTTTGGATGGAGCAATtcaacacACAAGAATGGTGGATTTCC GAGAATGGTGGATTTCC 429..438 TCAACACACA 139CTGACTTTGGATGGAGCAATGAGA 317 CTGACTTTGGATGGAGCAATtcaacacacaG (395)ATGGTGGATTTCCATA AGAATGGTGGATTTCCATA 429..439 TCAACACACA 140CTGACTTTGGATGGAGCAATAGAA 318 CTGACTTTGGATGGAGCAATtcaacacacag G (396)TGGTGGATTTCCATAC AGAATGGTGGATTTCCATAC 430..436 CAACACA 141TGACTTTGGATGGAGCAATTCAGA 319 TGACTTTGGATGGAGCAATTcaacacaCAGGAATGGTGGATTTCCA AGAATGGTGGATTTCCA 431..431 A 142GACTTTGGATGGAGCAATTCACAC 320 GACTTTGGATGGAGCAATTCaACACACAACAGAGAATGGTGGAT GAGAATGGTGGAT 431..432 AA 143 GACTTTGGATGGAGCAATTCCACA321 GACTTTGGATGGAGCAATTCaaCACACA CAGAGAATGGTGGATT GAGAATGGTGGATT431..438 AACACACA 144 GACTTTGGATGGAGCAATTCGAGA 322GACTTTGGATGGAGCAATTCaacacacaGA ATGGTGGATTTCCATA GAATGGTGGATTTCCATA431..441 AACACACAGA 145 GACTTTGGATGGAGCAATTCAATG 323GACTTTGGATGGAGCAATTCaacacacagag G (397) GTGGATTTCCATACACAATGGTGGATTTCCATACAC 432..433 AC 146 ACTTTGGATGGAGCAATTCAACAC 324ACTTTGGATGGAGCAATTCAacACACAG AGAGAATGGTGGATTT AGAATGGTGGATTT 432..435ACAC 147 ACTTTGGATGGAGCAATTCAACAG 325 ACTTTGGATGGAGCAATTCAacacACAGAAGAATGGTGGATTTCC GAATGGTGGATTTCC 432..437 ACACAC 148ACTTTGGATGGAGCAATTCAAGAG 326 ACTTTGGATGGAGCAATTCAacacacAGAAATGGTGGATTTCCAT GAATGGTGGATTTCCAT 432..439 ACACACAG 149ACTTTGGATGGAGCAATTCAAGAA 327 ACTTTGGATGGAGCAATTCAacacacagAGTGGTGGATTTCCATAC AATGGTGGATTTCCATAC 432...441 ACACACAGAG 150ACTTTGGATGGAGCAATTCAAATG 328 ACTTTGGATGGAGCAATTCAacacacagagA (398)GTGGATTTCCATACAC ATGGTGGATTTCCATACAC 432..446 ACACACAGAG 151ACTTTGGATGGAGCAATTCATGGA 329 ACTTTGGATGGAGCAATTCAacacacagaga AATGG (399)TTTCCATACACTGAAA atggTGGATTTCCATACACTGAAA 432..448 ACACACAGAG 152ACTTTGGATGGAGCAATTCAGATT 330 ACTTTGGATGGAGCAATTCAacacacagaga AATGGTGTCCATACACTGAAATG atggtgGATTTCCATACACTGAAATG (400) 433..433 C 153CTTTGGATGGAGCAATTCAAACAC 331 CTTTGGATGGAGCAATTCAAcACACAGAGAGAATGGTGGATTT AGAATGGTGGATTT 433..436 CACA 154CTTTGGATGGAGCAATTCAACAGA 332 CTTTGGATGGAGCAATTCAAcacaCAGAGGAATGGTGGATTTCCA AATGGTGGATTTCCA 433..437 CACAC 155CTTTGGATGGAGCAATTCAAAGAG 333 CTTTGGATGGAGCAATTCAAcacacAGAGAATGGTGGATTTCCAT AATGGTGGATTTCCAT 433..439 CACACAG 156CTTTGGATGGAGCAATTCAAAGAA 334 CTTTGGATGGAGCAATTCAAcacacagAGATGGTGGATTTCCATAC ATGGTGGATTTCCATAC 434..435 AC 157TTTGGATGGAGCAATTCAACACAG 335 TTTGGATGGAGCAATTCAACacACAGAGAGAATGGTGGATTTCC AATGGTGGATTTCC 434..437 ACAC 158TTTGGATGGAGCAATTCAACAGAG 336 TTTGGATGGAGCAATTCAACacacAGAGAATGGTGGATTTCCAT AATGGTGGATTTCCAT 434..438 ACACA 159TTTGGATGGAGCAATTCAACGAGA 337 TTTGGATGGAGCAATTCAACacacaGAGAATGGTGGATTTCCATA ATGGTGGATTTCCATA 435..436 CA 160TTGGATGGAGCAATTCAACACAGA 338 TTGGATGGAGCAATTCAACAcaCAGAGAGAATGGTGGATTTCCA ATGGTGGATTTCCA 435..438 CACA 161TTGGATGGAGCAATTCAACAGAGA 339 TTGGATGGAGCAATTCAACAcacaGAGAATGGTGGATTTCCATA ATGGTGGATTTCCATA 435..439 CACAG 162TTGGATGGAGCAATTCAACAAGAA 340 TTGGATGGAGCAATTCAACAcacagAGAATGGTGGATTTCCATAC TGGTGGATTTCCATAC 435..447 CACAGAGAAT 163TTGGATGGAGCAATTCAACAGGAT 341 TTGGATGGAGCAATTCAACAcacagagaatg GGT (401)TTCCATACACTGAAAT gtGGATTTCCATACACTGAAAT 436..437 AC 164TGGATGGAGCAATTCAACACAGAG 342 TGGATGGAGCAATTCAACACacAGAGAAATGGTGGATTTCCAT ATGGTGGATTTCCAT 436..439 ACAG 165TGGATGGAGCAATTCAACACAGAA 343 TGGATGGAGCAATTCAACACacagAGAATGGTGGATTTCCATAC TGGTGGATTTCCATAC 436..443 ACAGAGAA 166TGGATGGAGCAATTCAACACTGGT 344 TGGATGGAGCAATTCAACACacagagaaTGGGATTTCCATACACTG GTGGATTTCCATACACTG 437..438 CA 167GGATGGAGCAATTCAACACAGAG 345 GGATGGAGCAATTCAACACAcaGAGAAAATGGTGGATTTCCATA TGGTGGATTTCCATA 440..442 AGA 168TGGAGCAATTCAACACACAGATGG 346 TGGAGCAATTCAACACACAGagaATGGTTGGATTTCCATACACT GGATTTCCATACACT 440..443 AGAA 169TGGAGCAATTCAACACACAGTGGT 347 TGGAGCAATTCAACACACAGagaaTGGTGGATTTCCATACACTG GGATTTCCATACACTG 441..441 G 170GGAGCAATTCAACACACAGAAATG 348 GGAGCAATTCAACACACAGAgAATGGTGTGGATTTCCATACAC GGATTTCCATACAC 445..449 GGTGG 171CAATTCAACACACAGAGAATATTT 349 CAATTCAACACACAGAGAATggtggATTTCCATACACTGAAATGA CCATACACTGAAATGA 451..463 TTTCCATACAC 172AACACACAGAGAATGGTGGAAAA 350 AACACACAGAGAATGGTGGAtttccatacact TG (402)TGATTGTTCATCTTCCA gAAATGATTGTTCATCTTCCA

TABLE 5E A list of exemplary mutant alleles obtained in the PMT4 gene.PMT4 Mutant Reference Allele Allele Deleted sequence sequence sequence(SEQ ID Mutant (SEQ ID Position (SEQ ID No.) No.) Allele Sequence No.)Reference Allele Sequence 131...131 A 173 CGGCACTTCCAAACACCAAACGGCCA 351CGGCACTTCCAAACACCAAAaCGGCCAC CCATAATGGCACTT CATAATGGCACTT 541..551ATTCAACACA 174 TTTTGACTTTGGATGGAGCAAGAGAAT 352TTTTGACTTTGGATGGAGCAattcaacacacA C (403) GGTGGATTTCCATGAGAATGGTGGATTTCCAT 543..554 TCAACACACA 175 TTGACTTTGGATGGAGCAATGAATGGT353 TTGACTTTGGATGGAGCAATtcaacacacaga GA (404) GGATTTCCATACAGAATGGTGGATTTCCATACA 544..550 CAACACA 176 TGACTTTGGATGGAGCAATTCAGAGA 354TGACTTTGGATGGAGCAATTcaacacaCAG ATGGTGGATTTCCA AGAATGGTGGATTTCCA 544..553CAACACACAG 177 TGACTTTGGATGGAGCAATTAGAATGG 355TGACTTTGGATGGAGCAATTcaacacacagA (405) TGGATTTCCATAC GAATGGTGGATTTCCATAC545..555 AACACACAGA 178 GACTTTGGATGGAGCAATTCAATGGTG 356GACTTTGGATGGAGCAATTCaacacacagag G (406) GATTTCCATACACAATGGTGGATTTCCATACAC 545..557 AACACACAGA 179 GACTTTGGATGGAGCAATTCTGGTGGA357 GACTTTGGATGGAGCAATTCaacacacagaga GAA (407) TTTCCATACACTGaTGGTGGATTTCCATACACTG 546..547 AC 180 ACTTTGGATGGAGCAATTCAACACAG 358ACTTTGGATGGAGCAATTCAacACACAG AGAATGGTGGATTT AGAATGGTGGATTT 546..549 ACAC181 ACTTTGGATGGAGCAATTCAACAGAG 359 ACTTTGGATGGAGCAATTCAacacACAGAAATGGTGGATTTCC GAATGGTGGATTTCC 546..551 ACACAC 182ACTTTGGATGGAGCAATTCAAGAGAA 360 ACTTTGGATGGAGCAATTCAacacacAGAGTGGTGGATTTCCAT AATGGTGGATTTCCAT 546..552 ACACACA 183ACTTTGGATGGAGCAATTCAGAGAAT 361 ACTTTGGATGGAGCAATTCAacacacaGAGGGTGGATTTCCATA AATGGTGGATTTCCATA 546..553 ACACACAG 184ACTTTGGATGGAGCAATTCAAGAATG 362 ACTTTGGATGGAGCAATTCAacacacagAGGTGGATTTCCATAC AATGGTGGATTTCCATAC 547..550 CACA 185CTTTGGATGGAGCAATTCAACAGAGA 363 CTTTGGATGGAGCAATTCAAcacaCAGAGATGGTGGATTTCCA AATGGTGGATTTCCA 547..551 CACAC 186CTTTGGATGGAGCAATTCAAAGAGAA 364 CTTTGGATGGAGCAATTCAAcacacAGAGTGGTGGATTTCCAT AATGGTGGATTTCCAT 548..549 AC 187TTTGGATGGAGCAATTCAACACAGAG 365 TTTGGATGGAGCAATTCAACacACAGAGAATGGTGGATTTCC AATGGTGGATTTCC 548..551 ACAC 188TTTGGATGGAGCAATTCAACAGAGAA 366 TTTGGATGGAGCAATTCAACacacAGAGATGGTGGATTTCCAT ATGGTGGATTTCCAT 548..552 ACACA 189TTTGGATGGAGCAATTCAACGAGAAT 367 TTTGGATGGAGCAATTCAACacacaGAGAGGTGGATTTCCATA ATGGTGGATTTCCATA 549..552 CACA 190TTGGATGGAGCAATTCAACAGAGAAT 368 TTGGATGGAGCAATTCAACAcacaGAGAAGGTGGATTTCCATA TGGTGGATTTCCATA 549..553 CACAG 191TTGGATGGAGCAATTCAACAAGAATG 369 TTGGATGGAGCAATTCAACAcacagAGAAGTGGATTTCCATAC TGGTGGATTTCCATAC 550..551 AC 192TGGATGGAGCAATTCAACACAGAGAA 370 TGGATGGAGCAATTCAACACacAGAGAATGGTGGATTTCCAT TGGTGGATTTCCAT 550..552 ACA 193TGGATGGAGCAATTCAACACGAGAAT 371 TGGATGGAGCAATTCAACACacaGAGAAGGTGGATTTCCATA TGGTGGATTTCCATA 550..553 ACAG 194TGGATGGAGCAATTCAACACAGAATG 372 TGGATGGAGCAATTCAACACacagAGAATGTGGATTTCCATAC GGTGGATTTCCATAC 550..556 ACAGAGA 195TGGATGGAGCAATTCAACACATGGTG 373 TGGATGGAGCAATTCAACACacagagaATGGATTTCCATACACT GTGGATTTCCATACACT 551..552 CA 196GGATGGAGCAATTCAACACAGAGAAT 374 GGATGGAGCAATTCAACACAcaGAGAATGGTGGATTTCCATA GGTGGATTTCCATA 554..554 A 197 TGGAGCAATTCAACACACAGGAATGG375 TGGAGCAATTCAACACACAGaGAATGGT TGGATTTCCATACA GGATTTCCATACA 558..563TGGTGG 198 GCAATTCAACACACAGAGAAATTTCC 376 GCAATTCAACACACAGAGAAtggtggATTTATACACTGAAATGA CCATACACTGAAATGA 565..566 TT 199AACACACAGAGAATGGTGGATCCATA 377 AACACACAGAGAATGGTGGAttTCCATACCACTGAAATGATTG ACTGAAATGATTG 569..572 CATA 200CACAGAGAATGGTGGATTTCCACTGA 378 CACAGAGAATGGTGGATTTCcataCACTGAATGATTGTTCATC AAATGATTGTTCATC

Example 3: Alkaloid Analysis of PMT Edited Lines

Genome edited tobacco plants along with controls are grown in 10″ potsin green house with 75 PPM fertilizer. At flowering stage, plants aretopped and 2 weeks post topping lamina samples were collected from 3, 4,5 leaves from top and alkaloid levels are measured (Tables 6A to 6C)using a method in accordance with CORESTA Method No 62, Determination ofNicotine in Tobacco and Tobacco Products by Gas ChromatographicAnalysis, February 2005, and those defined in the Centers for DiseaseControl and Prevention's Protocol for Analysis of Nicotine, TotalMoisture and pH in Smokeless Tobacco Products, as published in theFederal Register Vol. 64, No. 55 Mar. 23, 1999 (and as amended in Vol.74, No. 4, Jan. 7, 2009).

Briefly, approximately 0.5 g of tobacco is extracted using liquid/liquidextraction into an organic solvent containing an internal standard andanalyzed by gas chromatography (GC) with flame ionization detection(FID). Results can be reported as weight percent (Wt %) on either an asis or dry weight basis. Reporting data on a dry weight basis requires anoven volatiles (OV) determination. Unless specified otherwise, total orindividual alkaloid levels or nicotine levels shown herein are on a dryweight basis (e.g., percent total alkaloid or percent nicotine).

Plants are also planted in the field, harvested, and tested foralkaloids and TSNA levels in cured tobacco. Both leaf yield and leafgrade are also assessed for PMT edited plants. Further, different mutantcombinations of individual PMT genes are generated and tested (e.g.,single, double, triple, or quadruple mutants).

Example 4: Comparing a Quintuple Pmt Knock-Out Mutant with OtherLow-Alkaloid Tobacco Plants

A quintuple pmt knock-out mutant line CS15 (see Table 4E for genotype,in the NLM (Ph Ph) background) is grown side by side with a PMT RNAitransgenic line (in the VA359 background, as described in US2015/0322451) and a low-nicotine KY171 (“LN KY171”) variety (the KY 171background harboring nic1 and nic2 double mutations). Leaves areharvested and cured via a dark fire curing method. Each line is analyzedfor nicotine and total alkaloid levels, leaf yield, and leaf quality(FIGS. 2 to 5). The data shows that suppressing PMT gene activity byediting all five PMT genes reduces nicotine level without comprisingleaf yield or quality.

Example 5: Obtaining Tobacco Lines with Edited Mutant Alleles in One orMore PMT Genes

Tobacco lines with mutations in individual PMT genes or selectedcombinations of PMT genes are obtained from the tobacco lines listed inTable 3. Crossing a quintuple, quadruple, triple, or double mutant(having mutations in five, four, three, or two PMT genes, respectively)to a non-mutated control line and selecting segregating progeny plantsfor specific PMT mutation combinations. Tables 7A to 7E representspossible mutant combinations being obtained. Each mutated gene can beeither homozygous or heterozygous for the mutation. Each of the mutantalleles listed in Tables 4A to 4E and Table 10 can be used to generatesingle, double, triple, quintuple, or quadruple mutants. Exemplaryindividual pmt mutant alleles are listed in Tables 9A to 9E.

Example 6: Further Reduction of Total Alkaloids by Combining PmtMutations with Mutations in Other Genes

To further reduce total alkaloids and/or selected individual alkaloids,pmt mutants are combined with mutations in additional genes related toalkaloid biosynthesis in tobacco, such as quinolate phosphoribosyltransferase (QPT) or quinolinate synthase (QS). Briefly, gene editing isused to mutant selected QPT and/or QS genes in a desired pmt mutantbackground (e.g., a quadruple or quintuple pmt mutant). In the resultingcombined qpt/pmt or qs/pmt mutants, alkaloids and TSNA levels are testedin cured tobacco. Both leaf yield and leaf grade are also assessed.

TABLE 6A Alkaloid levels in PMT edited lines in K326 (shown here andTables 6B, 6C, and 7 as weight percentage per gram leaf lamina (dryweight)) % Alkaloids Variety Plant ID Nicotine Total Alkaloids K32617GH1811 1.17 1.23 Control 17GH1822 1.63 1.71 17GH1806 1.69 1.7617GH1899 1.7893 1.9194 17GH1812 1.91 2 17GH1900 2.088 2.239 17GH18212.16 2.26 17GH1896 2.6006 2.7359 K326 17GH1810 0.0013 0.3 Edited17GH1808 0.0044 0.24 17GH1901 0.006 0.6958 17GH1893 0.0072 0.735117GH1804 0.0078 0.44 17GH1902 0.008 0.6245 18GH4 0.0102 0.2688 17GH18920.0209 0.1281

TABLE 6B Alkaloid levels in PMT edited lines in TN90 % Alkaloids TotalVariety Plant ID Nicotine Alkaloids TN90 17GH1838 1.88 1.98 Control17GH1923 2.0868 2.2136 17GH1924 2.2099 2.3394 17GH1718 2.29 2.4217GH1839 2.6 2.74 17GH1909 2.7639 2.9429 17GH1910 2.9346 3.1283 TN9017GH1699 0.0011 0.58 Edited 17GH1708 0.0014 0.56 17GH1847 0.0016 0.617GH1848 0.0018 0.42 17GH1724 0.0022 0.59 17GH1846 0.0022 0.41 17GH17220.0023 0.62 17GH1725 0.003 0.69 17GH1717 0.0035 0.7 17GH1719 0.0042 0.7517GH1845 0.0047 0.45 17GH1943 0.007 0.3464 18GH47 0.0072 1.0455 17GH19440.0074 0.403 17GH1932 0.0074 0.4758 17GH1936 0.0074 1.4394 17GH19180.0075 0.458 17GH1912 0.0078 0.5234 18GH31 0.0079 1.0902 18GH28 0.0080.8748 17GH1928 0.0081 1.1024 17GH1933 0.0083 0.6517 17GH1911 0.00880.281

TABLE 6C Alkaloid levels in PMT edited lines in Narrow Leaf Madole (NLM)% Alkaloids Total Variety Plant ID Nicotine Alkaloids NLM Control18GH126 2.0844 2.1734 18GH7 3.3504 3.5136 NLM Edited 18GH10 0.001 1.1418GH9 0.0012 0.92 18GH6 0.0019 1.46 18GH8 0.0022 1.46 17GH1905 0.00321.49 18GH5 0.0038 0.92 18GH130 0.0041 0.8756 18GH132 0.0044 0.633518GH79 0.0045 0.6182 18GH69 0.0069 0.7495 18GH71 0.007 0.7726 18GH1310.0077 0.4289 18GH66 0.0081 0.6951 18GH227 0.0086 0.8726 18GH78 0.00860.662 18GH72 0.0087 1.0048 18GH216 0.0089 1.2758 18GH65 0.0094 0.7018

TABLE 7 Relative changes in nicotine and total alkaloid levels inquintuple pmt knock-out mutants in various varieties. Average percentlevels of nicotine and total alkaloids are calculated based on percentlevel data from individual lines as shown in Tables 6A to 6C. Relativechanges reflect the nicotine or total alkaloid level in a quintuple pmtmutant relative to its control. Total Nicotine Alkaloids K326 Control1.880 1.982 K326 quintuple pmt 0.008 0.429 mutant Relative change 0.44%21.65% TN90 Control 2.395 2.538 TN90 quintuple pmt 0.005 0.655 mutantRelative change 0.22% 25.80% NLM Control 2.717 2.844 NLM quintuple pmt0.006 0.927 mutant Relative change 0.20% 32.59%

TABLE 8A A list of mutants obtained with various genotypic combinationsfor five PMT genes: single gene mutations PMT1a PMT1b PMT2 PMT3 PMT4 1Mutant WT WT WT WT 2 WT Mutant WT WT WT 3 WT WT Mutant WT WT 4 WT WT WTMutant WT 5 WT WT WT WT Mutant

TABLE 8B A list of mutants obtained with various genotypic combinationsfor five PMT genes: double gene mutations PMT1a PMT1b PMT2 PMT3 PMT4 1Mutant Mutant WT WT WT 2 Mutant WT Mutant WT WT 3 Mutant WT WT Mutant WT4 Mutant WT WT WT Mutant 5 WT Mutant Mutant WT WT 6 WT Mutant WT MutantWT 7 WT Mutant WT WT Mutant 8 WT WT Mutant Mutant WT 9 WT WT Mutant WTMutant 10 WT WT WT Mutant Mutant

TABLE 8C A list of mutants obtained with various genotypic combinationsfor five PMT genes: triple gene combinations PMT1a PMT1b PMT2 PMT3 PMT41 Mutant Mutant Mutant WT WT 2 Mutant Mutant WT Mutant WT 3 MutantMutant WT WT Mutant 4 Mutant WT Mutant Mutant WT 5 Mutant WT Mutant WTMutant 6 Mutant WT WT Mutant Mutant 7 WT Mutant Mutant Mutant WT 8 WTMutant Mutant WT Mutant 9 WT WT Mutant Mutant Mutant 10 WT Mutant MutantWT Mutant

TABLE 8D A list of mutants obtained with various genotypic combinationsfor five PMT genes: quadruple gene combinations PMT1a PMT1b PMT2 PMT3PMT4 1 Mutant Mutant Mutant Mutant WT 2 WT Mutant Mutant Mutant Mutant 3Mutant WT Mutant Mutant Mutant 4 Mutant Mutant WT Mutant Mutant 5 MutantMutant Mutant WT Mutant

TABLE 8E A list of mutants obtained with various genotypic combinationsfor five PMT genes: quintuple gene combinations PMT1a PMT1b PMT2 PMT3PMT4 1 Mutant Mutant Mutant Mutant Mutant

Example 7: PMT Genome Editing and Tobacco Line Development

Additional PMT knockout mutants are produced by editing all five PMTgenes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco lines.Tobacco protoplasts are transfected using polyethylene glycol (PEG) withplasmids encoding a genome editing technology (GET2) protein andspecific guide RNAs (gRNAs) targeting PMT genes at desired positions.Table 9 lists gRNA sequences used for PMT editing. Some gRNAs (e.g.,Nos. 6 and 7) are pooled together for targeting multiple PMT genes in asingle transfection.

TABLE 9 Guide RNAs for GET2 used in Example 7. ″Y″indicates that a gRNA is capable of targeting that PMT gene, while ″—″represents that a gRNA does not target that PMT gene. gRNA SequencePMT1a PMT1b PMT2 PMT3 PMT4 GATGGAGCAATTCAACATACAGA Y Y — — —(SEQ ID NO: 730) GATGGAGCAATTCAACACACAGA — — Y Y Y (SEQ ID NO: 731)

Transfected protoplasts are then immobilized in 1% agarose bead andsubjected to tissue culture. When calli grow up to ˜1 mm in diameter,they are spread on TOM2 plates. Calli are screened for insertions ordeletions (indels) at the target positions using fragment analysis.Candidates, showing size shifts compared to wildtype control, areselected for further culture and the consequent shoots are tested byfragment analysis again to confirm the presence of indels. Rooted shootsare potted and sequenced for the target positions to determine the exactsequences deleted. Young leaf from each plant is harvested and PCRamplified for PMT fragments using phirekit. PMT Libraries for each lineis indexed and 384 lines are pooled and sequenced using Miseq.

SNP analysis is carried out to determine both the exact edited pmtmutant allele sequences and the zygosity state at each PMT gene locus.Table 10 provides indels sequence information in each edited line ofvarious tobacco varieties (e.g., Basma, K326, Katerini, TN90, Izmir).

TABLE 10Mutant pmt alleles in various lines produced by genome editing using GET2. The position of each edited site (e.g., indels) is relativeto the nucleotide number on the corresponding cDNA sequence of each PMT gene (e.g., SEQ ID NO: 6 for PMT1a; SEQ ID NO: 7 for PMT1b; SEQID NO: 8 for PMT2; SEQ ID NO: 9 for PMT3; SEQ ID NO: 10 for PMT4). SEQ ID Numbers are assigned and shown for sequences of more than10 nucleotides. PMT1a PMT1b PMT2 PMT3 PMT4 Geno- Deleted Deleted DeletedDeleted Deleted type Line Sequence Position Sequence Position SequencePosition Sequence Position Sequence Position BASMA CS107 CAACAT 412.418ACAT 414..417 AC 348..349 ACAC 432..435 ACAC 546..549 A BASMA CS106 ACAT414..417 ACAT 414..417 AC 348..349 ACAC 432..435 ACAC 546..549 K326CS115 TCAACAT 411..420 ACAT 414..417 AC 348..349 CACAC 433..437 ACACA548..552 ACA (SEQ  ID NO: 379) K326 18GH ACAT 414..417 ACAT 414..417ACACAC 348..355 AC 432..433 ACACA 548..552 2162 AG K326 CS111 ACAT414..417 ACAT 414..417 AC 348..349 ACAC 432..435 CACAC 547..551 K326CS112 CATACA 415..421 AC 418..419 AC 348..349 ACAC 432..435 CACAC547..551 G K326 17GH CATACA 415..421 AC 418..419 AC 348..349 ACAC432..435 ACAC 546..549 1678- G 60 K326 CS131 ACAT 414..417 ACAT 414..417ACAC 348..351 AACACACA 431..439 ACAC 546..549 G KATERINI CS164 AT416..417 CAACAT 412..418 AC 348..349 ACAC 432..435 AC 546..547 AKATERINI CS163 ACAT 414..417 AT 416..417 AC 348..349 ACAC 432..435 AC546..547 KATERINI CS146 GAGCAA 404..422 ACAT 414..417 AC 348..349CAACACA 430..436 AC 546..547 TTCAACA TACAGA (SEQ ID NO: 408) KATERINICS147 ACAT 414..417 CAACAT 412..418 AC 348..349 CAACACA 430..436 AC546..547 A KATERINI CS150 AT 416..417 ACAT 414..417 AC 348..349 CACACAG433..439 AC 546..547 KATERINI CS151 AT 416..417 ACAT 414..417 AC348..349 ACAC 432..435 CAACAC 544..553 ACAG (SEQ ID NO: 390) KATERINICS148 ACAT 414..417 ACAT 414..417 AC 348..349 CACACAG 433..439 AC546..547 KATERINI CS149 ACAT 414..417 ACAT 414..417 AC 348..349 ACAC432..435 CAACAC 544..553 ACAG (SEQ ID NO: 390) KATERINI CS152 AC418..419 ACAT 414..417 AC 348..349 ACAC 432..435 ACAC 546..549 KATERINICS153 CAACAT 412.418 AC 414..415 AC 348..349 ACAC 432..435 ACAC 546..549A KATERINI CS102 ACAT 414..417 AACAT 413..417 ACACAC 348..355 AC432..433 AC 546..547 AG KATERINI CS103 AC 418..419 CAACAT 412. .418ACACAC 348..355 AC 432..433 AC 546..547 A AG TN90 CS143 TACAGA 417..423ACAT 414..417 AC 348..349 AC 432..433 ACAC 546..549 G TN90 18GH AC418..419 ACAT 414..417 ACAC 348..351 ACAC 432..435 AC 546..547 2169 TN90CS120 ACAT 414..417 ACAG 418..421 ACAC 348..351 AC 432..433 AC 546..547TN90 17GH ACAT 414..417 ACAT 414..417 AC 348..349 ACAG 436..439 AC546..547 1698- 22 TN90 17GH ACAT 414..417 AACAT 413..417 AC 348..349 AC432..433 ACAC 546..549 1700- 13 TN90 17GH ACAT 414..417 ACAT 414..417 AC348..349 AC 432..433 CACAG 549..553 1702- 17 TN90 18GH ACAT 414..417ACAT 414..417 ACAC 348..351 CAACACA 430..436 AC 546..547 2171 TN90 CS165ACAT 414..417 ACAT 414..417 ACAC 348..351 ACAC 432..435 ACAC 546..549TN90 CS118 ACAT 414..417 ACAT 414..417 AC 348..349 AC 432..433 ACAC546..549 TN90 CS133 GGAGCA 403..415 CAACAT 412..421 ACAC 348..351 AC432..433 AC 546..547 ATTCAAC ACAG (SEQ ID (SEQ ID NO: 409) NO: 380) TN9017GH CA 415..416 ACAT 414..417 ACAC 348..351 AC 432..433 AC 546..5471737- 24 IZMIR 18GH CAACAT 412..418 ATAGAG 416..417 ACAC 348..351 ACAC432..435 ACACAC 546..553 2254- A AA & AG 7 420..425

TABLE 11 provides the length (in nucleotides) of each PMT indel for eachgene in each line as provided in Table 10. Table 11. The length (innucleotides) of each indel for selected lines provided in Table 10.Genotype Line Seed Ids PMT1a PMT1b PMT2 PMT3 PMT4 BASMA CS107 CS107 7 42 4 4 BASMA CS106 CS106 4 4 2 4 4 K326 CS115 CS115 10 4 2 5 5 K32617GH1809-13 18GH2162 4 4 8 2 5 K326 CS111 CS111 4 4 2 4 5 K326 CS112CS112 7 2 2 4 5 K326 17GH1678-60 17GH1678-60 7 2 2 4 4 K326 CS131 CS1314 4 4 9 4 KATERINI 18GH709-01 CS164 2 7 2 4 2 KATERINI 18GH709-08 CS1634 2 2 4 2 KATERINI 18GH414-11 CS146 19 4 2 7 2 KATERINI 18GH414-19 CS1474 7 2 7 2 KATERINI 18GH437-04 CS150 2 4 2 7 2 KATERINI 18GH437-08 CS1512 4 2 4 10 KATERINI 18GH437-32 CS148 4 4 2 7 2 KATERINI 18GH437-39 CS1494 4 2 4 10 KATERINI 18GH449-26 CS152 2 4 2 4 4 KATERINI 18GH449-33 CS1537 2 2 4 4 KATERINI 18GH125-48 CS162 2 7 8 2 2 KATERINI CS102 CS102 4 5 82 2 KATERINI CS103 CS103 2 7 8 2 2 TN90 17GH1719-30 CS143 7 4 2 2 4 TN9017GH1740-36 18GH2169 2 4 4 4 2 TN90 17GH1698-22 17GH1698-22 4 4 2 4 2TN90 17GH1700-13 17GH1700-13 4 5 2 2 4 TN90 17GH1702-17 17GH1702-17 4 42 2 5 TN90 17GH1849-01 18GH2171 4 4 4 7 2 TN90 17GH1849-48 CS165 4 4 4 44 TN90 17GH1737-24 17GH1737-24 2 4 4 2 2 TN90 CS118 CS118 4 4 2 2 4 TN90CS133 CS133 13 10 4 2 2 TN90 CS120 CS120 4 4 4 2 2 IZMIR 18GH1108-0718GH2254-7 7 8 4 4 8

Tables 12A to 12E provide genomic sequences of approximately 90nucleotides from each pmt mutant allele with the edited site in themiddle of the genomic sequence (e.g., 45 nucleotides on each side of thedeleted or inserted sequence site).

TABLE 12AA list of exemplary mutant alleles obtained in the PMT1a gene. Mutant allele sequences listed here represent approximately90-nucleotide-long genomic sequences from each edited PMT1a gene with the edited site in the middle of thegenomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant allele corresponds to theindel provided for each line in Table 10. The lowercase letters in the reference allele sequence (SEQ ID NO: 6) denotewhich nucleotides are deleted in the mutant allele. ReferenceMutant Allele Allele SEQ Genotype Line Mutant Allele Sequence SEQ ID NO.Reference Allele Sequence ID NO. BASMA CS107 TCAGCAACTTATGGGAAGGTTCTGACT410 TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 442TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA BASMA CS106TCAGCAACTTATGGGAAGGTTCTGACT 411TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 443TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS115TCAGCAACTTATGGGAAGGTTCTGACT 412TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATtca 444TTGGATGGAGCAATGAGAATGGTGGATacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTCCATACACTGAAATGATTGTTCATCT TCTA A K326 18GH2162TCAGCAACTTATGGGAAGGTTCTGACT 413TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 445TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS111TCAGCAACTTATGGGAAGGTTCTGACT 414TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 446TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS112TCAGCAACTTATGGGAAGGTTCTGACT 415TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 447TTGGATGGAGCAATTCAAAGAATGGTGAAcatacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTATCAAAGAATGG ATCTA K326 17GH1678-TCAGCAACTTATGGGAAGGTTCTGACT 416TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 448 60TTGGATGGAGCAATTCAAAGAATGGTGAAcatacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTATCAAAGAATGG ATCTA K326 CS131TCAGCAACTTATGGGAAGGTTCTGACT 417TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 449TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS164TCAGCAACTTATGGGAAGGTTCTGACT 418TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 450TTGGATGGAGCAATTCAACACAGAGAA AACatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS163TCAGCAACTTATGGGAAGGTTCTGACT 419TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 451TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS146TCAGCAACTTATGGGAAGGTTCTGACT 420TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAgcaattcaaca 452TTGGATGGAATGGTGGATTTCCATACAtacagagaATGGTGGATTTCCATACACTGAAATGATTGTTCATCTA CTGAAATGATTGTTCATCTAKATERINI CS147 TCAGCAACTTATGGGAAGGTTCTGACT 421TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 453TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS150TCAGCAACTTATGGGAAGGTTCTGACT 422TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 454TTGGATGGAGCAATTCAACACAGAGAA AACatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS151TCAGCAACTTATGGGAAGGTTCTGACT 423TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 455TTGGATGGAGCAATTCAACACAGAGAA AACatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS148TCAGCAACTTATGGGAAGGTTCTGACT 424TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 456TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS149TCAGCAACTTATGGGAAGGTTCTGACT 425TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 457TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS152TCAGCAACTTATGGGAAGGTTCTGACT 426TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 458TTGGATGGAGCAATTCAACATAGAGAA AACATacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS153TCAGCAACTTATGGGAAGGTTCTGACT 427TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 459TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA KATERINI CS102TCAGCAACTTATGGGAAGGTTCTGACT 428TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 460TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS103TCAGCAACTTATGGGAAGGTTCTGACT 429TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 461TTGGATGGAGCAATTCAACATAGAGAA AACATacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA TN90 CS143TCAGCAACTTATGGGAAGGTTCTGACT 430TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 462TTGGATGGAGCAATTCAACAAATGGTGAACAtacagagAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA TN90 18GH2169TCAGCAACTTATGGGAAGGTTCTGACT 431TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 463TTGGATGGAGCAATTCAACATAGAGAA AACATacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA TN90 CS120TCAGCAACTTATGGGAAGGTTCTGACT 432TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 464TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1698-TCAGCAACTTATGGGAAGGTTCTGACT 433TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 465 22TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1700-TCAGCAACTTATGGGAAGGTTCTGACT 434TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 466 13TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1702-TCAGCAACTTATGGGAAGGTTCTGACT 435TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 467 17TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 18GH2171TCAGCAACTTATGGGAAGGTTCTGACT 436TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 468TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 CS165TCAGCAACTTATGGGAAGGTTCTGACT 437TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 469TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 CS118TCAGCAACTTATGGGAAGGTTCTGACT 438TCAGCAACTTATGGGAAGGTTCTGACTTTGGATggagcaattcaacAT 470TTGGATATACAGAGAATGGTGGATTTC ACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATCCATACACTGAAATGATTGTTCATCTA TA TN90 CS113 TCAGCAACTTATGGGAAGGTTCTGACT 439TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 471TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1737-TCAGCAACTTATGGGAAGGTTCTGACT 440TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 472 24TTGGATGGAGCAATTCAATACAGAGAA AAcaTACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA IZMIR 18GH2254-7TCAGCAACTTATGGGAAGGTTCTGACT 441TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 473TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA

TABLE 12BA list of exemplary mutant alleles obtained in the PMT1b gene. Mutant allele sequences listed here represent approximately 90-nucleotide-long genomic sequences from each edited PMT1b gene with the edited site in the middle of the genomic sequence (e.g., 45 nucleotides oneach side of the deleted sequence site). The mutant allele corresponds to the indel provided for each line in Table 10. The lowercaseletters in the reference allele sequence (SEQ ID NO: 7) denote which nucleotides are deleted in the mutant allele.Reference Mutant Allele Allele SEQ Genotype Line Mutant Allele SequenceSEQ ID NO. Reference Allele Sequence ID NO. BASMA CS107TCAGCAACTTATGGGAAGGTTCTGACT 474TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 506TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA BASMA CS106TCAGCAACTTATGGGAAGGTTCTGACT 475TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 507TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS115TCAGCAACTTATGGGAAGGTTCTGACT 476TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 508TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 18GH2162TCAGCAACTTATGGGAAGGTTCTGACT 477TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 509TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS111TCAGCAACTTATGGGAAGGTTCTGACT 478TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 510TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA K326 CS112TCAGCAACTTATGGGAAGGTTCTGACT 479TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 511TTGGATGGAGCAATTCAACATAGAGAA AACATacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA K326 17GH1678-TCAGCAACTTATGGGAAGGTTCTGACT 480TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 512 60TTGGATGGAGCAATTCAACATAGAGAA AACATacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA K326 CS131TCAGCAACTTATGGGAAGGTTCTGACT 481TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 513TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS164TCAGCAACTTATGGGAAGGTTCTGACT 482TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 514TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA KATERINI CS163TCAGCAACTTATGGGAAGGTTCTGACT 483TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 515TTGGATGGAGCAATTCAACACAGAGAA AACatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS146TCAGCAACTTATGGGAAGGTTCTGACT 484TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 516TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS147TCAGCAACTTATGGGAAGGTTCTGACT 485TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 517TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA KATERINI CS150TCAGCAACTTATGGGAAGGTTCTGACT 486TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 518TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS151TCAGCAACTTATGGGAAGGTTCTGACT 487TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 519TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS148TCAGCAACTTATGGGAAGGTTCTGACT 488TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 520TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS149TCAGCAACTTATGGGAAGGTTCTGACT 489TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 521TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS152TCAGCAACTTATGGGAAGGTTCTGACT 490TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 522TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA KATERINI CS153TCAGCAACTTATGGGAAGGTTCTGACT 491TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 523TTGGATGGAGCAATTCAATACAGAGAA AAcaTACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTA TGTTCATCTA KATERINI CS102TCAGCAACTTATGGGAAGGTTCTGACT 492TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 524TTGGATGGAGCAATTCACAGAGAATGGAacataCAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCTGGATTTCCATACACTGAAATGATTGT ATCTA TCATCTA KATERINI CS103TCAGCAACTTATGGGAAGGTTCTGACT 493TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 525TTGGATGGAGCAATTCAGAGAATGGTGAacatacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTA ATCTA TN90 CS143TCAGCAACTTATGGGAAGGTTCTGACT 494TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 526TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 18GH2169TCAGCAACTTATGGGAAGGTTCTGACT 495TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 527TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 CS120TCAGCAACTTATGGGAAGGTTCTGACT 496TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 528TTGGATGGAGCAATTCAACATAGAATG AACATacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1698-TCAGCAACTTATGGGAAGGTTCTGACT 497TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 529 22TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1700-TCAGCAACTTATGGGAAGGTTCTGACT 498TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 530 13TTGGATGGAGCAATTCACAGAGAATGGAacataCAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCTGGATTTCCATACACTGAAATGATTGT ATCTA TCATCTA TN90 17GH1702-TCAGCAACTTATGGGAAGGTTCTGACT 499TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 531 17TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 18GH2171TCAGCAACTTATGGGAAGGTTCTGACT 500TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 532TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 CS165TCAGCAACTTATGGGAAGGTTCTGACT 501TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 533TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 CS118TCAGCAACTTATGGGAAGGTTCTGACT 502TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTc 534TTGGATGGAGCAATTAGAATGGTGGATaacatacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTCCATACACTGAAATGATTGTTCATCT TCTA A TN90 CS113TCAGCAACTTATGGGAAGGTTCTGACT 503TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 535TTGGATGGAGCAATTCAACATAGAATG AACATacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA TN90 17GH1737-TCAGCAACTTATGGGAAGGTTCTGACT 504TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 536 24TTGGATGGAGCAATTCAACAGAGAATG AacatACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTA TTCATCTA IZMIR 18GH2254-7TCAGCAACTTATGGGAAGGTTCTGACT 505TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 537TTGGATGGAGCAATTCAACACTGGTGGAAcatacagagACTGGTGGATTTCCATACACTGAAATGATTGTTCAATTTCCATACACTGAAATGATTGTTCAT TCTA CTA

TABLE 12CA list of exemplary mutant alleles obtained in the PMT2 gene. Mutant allele sequences listed here represent approximately 90-nucleotide-long genomic sequences from each edited PMT2 gene with the edited site in the middle of the genomic sequence (e.g., 45 nucleotides oneach side of the deleted sequence site). The mutant allele corresponds to the indel provided for each line in Table 10. The lowercaseletters in the reference allele sequence (SEQ ID NO: 8) denote which nucleotides are deleted in the mutant allele.Reference Mutant Allele Allele SEQ Genotype Line Mutant Allele SequenceSEQ ID NO. Reference Allele Sequence ID NO. BASMA CS107TCAGCAACTTATGGGAAGGTTCTGACT 538TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 570TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT BASMA CS106TCAGCAACTTATGGGAAGGTTCTGACT 539TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 571TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 CS115TCAGCAACTTATGGGAAGGTTCTGACT 540TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 572TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 18GH2162TCAGCAACTTATGGGAAGGTTCTGACT 541TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 573TTGGATGGAGCAATTCAAGAATGGTGGAacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTTCCATACACTGAAATGATTGTTCAT ATCTT CTT K326 CS111TCAGCAACTTATGGGAAGGTTCTGACT 542TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 574TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 CS112TCAGCAACTTATGGGAAGGTTCTGACT 543TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 575TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 17GH1678-TCAGCAACTTATGGGAAGGTTCTGACT 544TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 576 60TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 CS131TCAGCAACTTATGGGAAGGTTCTGACT 545TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 577TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS164TCAGCAACTTATGGGAAGGTTCTGACT 546TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 578TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS163TCAGCAACTTATGGGAAGGTTCTGACT 547TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 579TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS146TCAGCAACTTATGGGAAGGTTCTGACT 548TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 580TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS147TCAGCAACTTATGGGAAGGTTCTGACT 549TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 581TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS150TCAGCAACTTATGGGAAGGTTCTGACT 550TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 582TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS151TCAGCAACTTATGGGAAGGTTCTGACT 551TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 583TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS148TCAGCAACTTATGGGAAGGTTCTGACT 552TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 584TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS149TCAGCAACTTATGGGAAGGTTCTGACT 553TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 585TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS152TCAGCAACTTATGGGAAGGTTCTGACT 554TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 586TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS153TCAGCAACTTATGGGAAGGTTCTGACT 555TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 587TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS102TCAGCAACTTATGGGAAGGTTCTGACT 556TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 588TTGGATGGAGCAATTCAAGAATGGTGGAacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTTCCATACACTGAAATGATTGTTCAT ATCTT CTT KATERINI CS103TCAGCAACTTATGGGAAGGTTCTGACT 557TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 589TTGGATGGAGCAATTCAAGAATGGTGGAacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTTCCATACACTGAAATGATTGTTCAT ATCTT CTT TN90 CS143TCAGCAACTTATGGGAAGGTTCTGACT 558TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 590TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 18GH2169TCAGCAACTTATGGGAAGGTTCTGACT 559TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 591TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS120TCAGCAACTTATGGGAAGGTTCTGACT 560TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 592TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 17GH1698-TCAGCAACTTATGGGAAGGTTCTGACT 561TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 593 22TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1700-TCAGCAACTTATGGGAAGGTTCTGACT 562TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 594 13TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1702-TCAGCAACTTATGGGAAGGTTCTGACT 563TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 595 17TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 18GH2171TCAGCAACTTATGGGAAGGTTCTGACT 564TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 596TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS165TCAGCAACTTATGGGAAGGTTCTGACT 565TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 597TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS118TCAGCAACTTATGGGAAGGTTCTGACT 566TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 598TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS113TCAGCAACTTATGGGAAGGTTCTGACT 567TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 599TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 17GH1737-TCAGCAACTTATGGGAAGGTTCTGACT 568TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 600 24TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT IZMIR 18GH2254-7TCAGCAACTTATGGGAAGGTTCTGACT 569TCAGCAACTTATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 601TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT

TABLE 12DA list of exemplary mutant alleles obtained in the PMT3 gene. Mutant allele sequences listed here represent approximately 90-nucleotide-long genomic sequences from each edited PMT3 gene with the edited site in the middle of the genomic sequence (e.g., 4nucleotides oneach side of the deleted sequence site). The mutant allele corresponds to the indel provided for each line in Table 10. The lowercaseletters in the reference allele sequence (SEQ ID NO: 9) denote which nucleotides are deleted in the mutant allele.Reference Mutant Allele Allele SEQ Genotype Line Mutant Allele SequenceSEQ ID NO. Reference Allele Sequence ID NO. BASMA CS107TCAGCAACATATGGGAAGGTTCTGACT 602TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 634TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT BASMA CS106TCAGCAACATATGGGAAGGTTCTGACT 603TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 635TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 CS115TCAGCAACATATGGGAAGGTTCTGACT 604TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 636TTGGATGGAGCAATTCAAAGAGAATGG AACacacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT K326 18GH2162TCAGCAACATATGGGAAGGTTCTGACT 605TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 637TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT K326 CS111TCAGCAACATATGGGAAGGTTCTGACT 606TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 638TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 CS112TCAGCAACATATGGGAAGGTTCTGACT 607TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 639TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 17GH1678-TCAGCAACATATGGGAAGGTTCTGACT 608TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 640 60TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 CS131TCAGCAACATATGGGAAGGTTCTGACT 609TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 641TTGGATGGAGCAATTCAGAATGGTGGAaacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTTCCATACACTGAAATGATTGTTCATC TCTT TT KATERINI CS164TCAGCAACATATGGGAAGGTTCTGACT 610TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 642TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS163TCAGCAACATATGGGAAGGTTCTGACT 611TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 643TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS146TCAGCAACATATGGGAAGGTTCTGACT 612TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTc 644TTGGATGGAGCAATTCAGAGAATGGTGaacacaCAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTT ATCTT KATERINI CS147TCAGCAACATATGGGAAGGTTCTGACT 613TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTc 645TTGGATGGAGCAATTCAGAGAATGGTGaacacaCAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTT ATCTT KATERINI CS150TCAGCAACATATGGGAAGGTTCTGACT 614TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 646TTGGATGGAGCAATTCAAAGAATGGTGAAcacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTT ATCTT KATERINI CS151TCAGCAACATATGGGAAGGTTCTGACT 615TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 647TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS148TCAGCAACATATGGGAAGGTTCTGACT 616TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 648TTGGATGGAGCAATTCAAAGAATGGTGAAcacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTT ATCTT KATERINI CS149TCAGCAACATATGGGAAGGTTCTGACT 617TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 649TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS152TCAGCAACATATGGGAAGGTTCTGACT 618TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 650TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS153TCAGCAACATATGGGAAGGTTCTGACT 619TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 651TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS102TCAGCAACATATGGGAAGGTTCTGACT 620TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 652TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS103TCAGCAACATATGGGAAGGTTCTGACT 621TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 653TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS143TCAGCAACATATGGGAAGGTTCTGACT 622TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 654TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 18GH2169TCAGCAACATATGGGAAGGTTCTGACT 623TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 655TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS120TCAGCAACATATGGGAAGGTTCTGACT 624TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 656TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1698-TCAGCAACATATGGGAAGGTTCTGACT 625TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 657 22TTGGATGGAGCAATTCAACACAGAATG AACACacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 17GH1700-TCAGCAACATATGGGAAGGTTCTGACT 626TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 658 13TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1702-TCAGCAACATATGGGAAGGTTCTGACT 627TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 659 17TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 18GH2171TCAGCAACATATGGGAAGGTTCTGACT 628TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTc 660TTGGATGGAGCAATTCAGAGAATGGTGaacacaCAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTCGATTTCCATACACTGAAATGATTGTTC ATCTT ATCTT TN90 CS165TCAGCAACATATGGGAAGGTTCTGACT 629TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 661TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS118TCAGCAACATATGGGAAGGTTCTGACT 630TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 662TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS113TCAGCAACATATGGGAAGGTTCTGACT 631TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 663TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1737-TCAGCAACATATGGGAAGGTTCTGACT 632TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 664 24TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT IZMIR 18GH2254-7TCAGCAACATATGGGAAGGTTCTGACT 633TCAGCAACATATGGGAAGGTTCTGACTTTGGATGGAGCAATTC 665TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT

TABLE 12EA list of exemplary mutant alleles obtained in the PMT4 gene. Mutant allele sequences listed here represent approximately 90-nucleotide-long genomic sequences from each edited PMT4 gene with the edited site in the middle of the genomic sequence (e.g., 45 nucleotides oneach side of the deleted sequence site). The mutant allele corresponds to the indel provided for each line in Table 10. The lowercaseletters in the reference allele sequence (SEQ ID NO: 10) denote which nucleotides are deleted in the mutant allele.Reference Mutant Allele Allele SEQ Genotype Line Mutant Allele SequenceSEQ ID NO. Reference Allele Sequence ID NO. BASMA CS107TCAGCAACATATGGGAAGGTTTTGACT 666TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 698TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT BASMA CS106TCAGCAACATATGGGAAGGTTTTGACT 667TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 699TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 CS115TCAGCAACATATGGGAAGGTTTTGACT 668TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 700TTGGATGGAGCAATTCAACGAGAATGG AACacacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT K326 18GH2162TCAGCAACATATGGGAAGGTTTTGACT 669TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 701TTGGATGGAGCAATTCAACGAGAATGG AACacacaGAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT K326 CS111TCAGCAACATATGGGAAGGTTTTGACT 670TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 702TTGGATGGAGCAATTCAAAGAGAATGG AAcacacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT K326 CS112TCAGCAACATATGGGAAGGTTTTGACT 671TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 703TTGGATGGAGCAATTCAAAGAGAATGG AAcacacAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT K326 17GH1678-TCAGCAACATATGGGAAGGTTTTGACT 672TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 704 60TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT K326 CS131TCAGCAACATATGGGAAGGTTTTGACT 673TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 705TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS164TCAGCAACATATGGGAAGGTTTTGACT 674TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 706TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS163TCAGCAACATATGGGAAGGTTTTGACT 675TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 707TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS146TCAGCAACATATGGGAAGGTTTTGACT 676TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 708TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS147TCAGCAACATATGGGAAGGTTTTGACT 677TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 709TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS150TCAGCAACATATGGGAAGGTTTTGACT 678TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 710TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS151TCAGCAACATATGGGAAGGTTTTGACT 679TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTc 711TTGGATGGAGCAATTAGAATGGTGGATaacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTCCATACACTGAAATGATTGTTCATCT TCTT T KATERINI CS148TCAGCAACATATGGGAAGGTTTTGACT 680TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 712TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS149TCAGCAACATATGGGAAGGTTTTGACT 681TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTc 713TTGGATGGAGCAATTAGAATGGTGGATaacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTCCATACACTGAAATGATTGTTCATCT TCTT T KATERINI CS152TCAGCAACATATGGGAAGGTTTTGACT 682TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 714TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS153TCAGCAACATATGGGAAGGTTTTGACT 683TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 715TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT KATERINI CS102TCAGCAACATATGGGAAGGTTTTGACT 684TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 716TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT KATERINI CS103TCAGCAACATATGGGAAGGTTTTGACT 685TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 717TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS143TCAGCAACATATGGGAAGGTTTTGACT 686TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 718TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 18GH2169TCAGCAACATATGGGAAGGTTTTGACT 687TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 719TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS120TCAGCAACATATGGGAAGGTTTTGACT 688TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 720TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1698-TCAGCAACATATGGGAAGGTTTTGACT 689TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 721 22TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1700-TCAGCAACATATGGGAAGGTTTTGACT 690TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 722 13TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 17GH1702-TCAGCAACATATGGGAAGGTTTTGACT 691TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 723 17TTGGATGGAGCAATTCAACAAGAATGG AACAcacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTTGGATTTCCATACACTGAAATGATTGT CATCTT TCATCTT TN90 18GH2171TCAGCAACATATGGGAAGGTTTTGACT 692TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 724TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS165TCAGCAACATATGGGAAGGTTTTGACT 693TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 725TTGGATGGAGCAATTCAACAGAGAATG AacacACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGTGGATTTCCATACACTGAAATGATTG CATCTT TTCATCTT TN90 CS118TCAGCAACATATGGGAAGGTTTTGACT 694TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 726TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 CS113TCAGCAACATATGGGAAGGTTTTGACT 695TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 727TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT TN90 17GH1737-TCAGCAACATATGGGAAGGTTTTGACT 696TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 728 24TTGGATGGAGCAATTCAACACAGAGAA AacACACAGAGAATGGTGGATTTCCATACACTGAAATGATTGTTGGTGGATTTCCATACACTGAAATGAT TCATCTT TGTTCATCTT IZMIR 18GH2254-7TCAGCAACATATGGGAAGGTTTTGACT 697TCAGCAACATATGGGAAGGTTTTGACTTTGGATGGAGCAATTC 729TTGGATGGAGCAATTCAAGAATGGTGGAacacacagAGAATGGTGGATTTCCATACACTGAAATGATTGTTCATTTCCATACACTGAAATGATTGTTCAT ATCTT CTT

Example 8. Alkaloid Analysis of PMT Edited Lines

Homozygous genome edited tobacco lines from Example 7, along withcontrol lines, are grown in a field. At flowering stage, plants aretopped and two-weeks post topping, lamina samples are collected from thethird, fourth, and fifth leaves from the top of the plant and alkaloidlevels are measured (see Tables 13A-13C) using a method in accordancewith CORESTA Method No 62, Determination of Nicotine in Tobacco andTobacco Products by Gas Chromatographic Analysis, February 2005, andthose defined in the Centers for Disease Control and Prevention'sProtocol for Analysis of Nicotine, Total Moisture and pH in SmokelessTobacco Products, as published in the Federal Register Vol. 64, No. 55Mar. 23, 1999 (and as amended in Vol. 74, No. 4, Jan. 7, 2009).

Approximately 0.5 g of tobacco is extracted using liquid/liquidextraction into an organic solvent containing an internal standard andanalyzed by gas chromatography (GC) with flame ionization detection(FID). Results can be reported as weight percent (Wt %) on either on asis or dry weight basis. Reporting data on a dry weight basis requires anoven volatiles (OV) determination. Unless specified otherwise, total orindividual alkaloid levels or nicotine levels shown herein are on a dryweight basis (e.g., percent total alkaloid or percent nicotine).

Plants are also planted in the field, harvested, and tested foralkaloids and TSNA levels in cured tobacco. Both leaf yield and leafgrade are also assessed for PMT edited plants.

TABLE 13A Nicotine analysis of K326 and TN90 PMT edited lines after twoweeks after flowering. Nicotine Variety Line Replicate (mg/g tissue) K326 CS111 1 0.023 2 0.024 CS131 1 0.022 2 0.018 3 0.021 CS115 1 0.023 20.015 Control 1 16.8 2 17.2 3 16.6 TN 90 CS116 1 0.029 LC 2 0.022 CS1331 0.016 2 0.018 CS135 1 0.027 2 0.031 CS120 1 0.022 2 0.045 CS137 1 0.072 0.048 Control 1 29.5 2 29.8 3 24.2

TABLE 13B Nicotine analysis of K326 and TN90 PMT edited lines aftertwo-weeks after topping. Nicotine Variety Group Replicate (mg/g tissue)K 326 CS111 1 0.024 2 0.025 CS131 1 0.021 2 0.02 3 0.019 CS115 1 0.018Control 1 16.771 2 17.212 3 16.581 4 22.734 TN 90 CS116 1 0.015 LC CS1331 0.036 2 0.018 CS135 1 0.018 2 0.019 CS120 1 0.04 2 0.025 CS137 1 0.0512 0.057 Control 1 29.472 2 29.776 3 24.22 4 24.939

TABLE 13C Nicotine analysis of Katerini and Basma PMT edited lines aftertwo-weeks after flowering. Nicotine Variety Group Replicate (mg/gtissue) Katerini CS102 1 0.032 2 0.028 CS103 1 0.017 Control 1 26.109 226.466 3 27.091 Basma CS107 1 0.029 CS108 1 0.014 2 0.018 Control 121.979 2 20.88 3 23.499

Example 9. Development of Male Sterile PMT Edited Lines

PMT edited hybrid lines are developed using the lines from Example 7.Hybrid lines are grown in the field and used as progenitors for malesterile lines. See Table 14.

TABLE 14 PMT edited very low nicotine male sterile lines Pollen sourceF1 hybrid seed Male Sterile Variety (line) (line) MS Katerini CS102dCS11 CS103 dCS12 MS Basma CS106 dCS13 CS107 dCS14 MS K326 CS111 dCS15CS115 dCS16 MS TN90 CS118 dCS17 CS120 dCS18 MS Izmir 18GH2254 dS2697

Example 10. PMT Edited Lines Resist Mold During Curing

Tobacco leaf harvested from several low alkaloid tobacco lines issubjected to standard air curing practices. The tobacco leaves areexamined for mold after the completion of curing.

Tobacco from the LA BU 21 exhibits more mold infestation than TN90 LC, aTN90 variety comprising an RNAi construct to downregulate all five PMTgenes, a TN90 variety comprising an RNAi construct to downregulate thealkaloid biosynthesis gene PR50, and four PMT edited lines (CS47, CS59,CS63, and CS64) in a TN90 genetic background. See Table 15 and FIGS. 6Ato 6E and 7.

TABLE 15 Mold damage exhibited by tobacco lines. Percentage of Mold MoldRating Significant Variety Replicate 1 Replicate 2 Replicate 3 Replicate4 Mold Some Mold Little/No Mold TN90 LC G G G G G G G G G G G G 0% 0%100% LA BU 21 G S G B S G S S S B S S 17% 58% 25% TN90 (PMT RNAi) G G GG G G G G G G G G 0% 0% 100% TN90 (PR50 RNai) G G G G G G G G G G G G 0%0% 100% CS47 G G G G G G G G G G G G 0% 0% 100% CS59 G G G G G G G G G GG G 0% 0% 100% CS63 G G G G G G G G G G G G 0% 0% 100% CS64 G G G G G GG G G S S G 0% 17% 83% “G” refers to little/no mold; “S” refers to somemold; and “B” refers to significant mold. Percentage of Mold refers tothe percentage of air cured sticks of tobacco exhibited each category ofmold damage.

1. A tobacco plant, or part thereof, comprising one or more mutantalleles in at least one PMT gene selected from the group consisting ofPMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein said tobacco plant iscapable of producing a leaf comprising a nicotine level less than thenicotine level of a leaf from a control tobacco plant not having saidone or more mutant alleles when grown and processed under comparableconditions.
 2. The tobacco plant, or part thereof, of claim 1, whereinsaid tobacco plant comprises one or more mutant alleles in at least twoPMT genes selected from the group consisting of PMT1a, PMT1b, PMT2,PMT3, and PMT4.
 3. The tobacco plant, or part thereof, of claim 1,wherein said tobacco plant comprises one or more mutant alleles in atleast three PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4.
 4. The tobacco plant, or part thereof, ofclaim 1, wherein said tobacco plant comprises one or more mutant allelesin at least four PMT genes selected from the group consisting of PMT1a,PMT1b, PMT2, PMT3, and PMT4.
 5. The tobacco plant, or part thereof, ofclaim 1, wherein said tobacco plant comprises one or more mutant allelesin five PMT genes selected from the group consisting of PMT1a, PMT1b,PMT2, PMT3, and PMT4.
 6. The tobacco plant, or part thereof, of any oneof claim 1, wherein said tobacco plant is capable of producing a leafcomprising a nicotine level less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of thenicotine level of a leaf from a control tobacco plant not having saidone or more mutant alleles when grown and processed under comparableconditions.
 7. The tobacco plant, or part thereof, of any one of claim1, wherein said tobacco plant is capable of producing a leaf comprisinga total alkaloid level less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of thetotal alkaloid level of a leaf from said control tobacco plant whengrown and processed under comparable conditions.
 8. (canceled)
 9. Thetobacco plant, or part thereof, of any one of claim 1, wherein said oneor more mutant alleles comprise a mutation in a sequence region selectedfrom the group consisting of a promoter, 5′ UTR, first exon, firstintron, second exon, second intron, third exon, 3′ UTR, terminator, andany combination thereof.
 10. The tobacco plant, or part thereof, of anyone of claim 1, wherein said one or more mutant alleles comprise one ormore mutation types selected from the group consisting of a nonsensemutation, a missense mutation, a frameshift mutation, a splice-sitemutation, and any combination thereof.
 11. The tobacco plant, or partthereof, of any one of claim 1 to 10, wherein said one or more mutantalleles result in one or more of the following: a PMT proteintruncation, a non-translatable PMT gene transcript, a non-functional PMTprotein, a premature stop codon in a PMT gene, and any combinationthereof.
 12. The tobacco plant, or part thereof, of any one of claim 1to 11 wherein said one or more mutant alleles comprise a mutationselected from the group consisting of a substitution, a deletion, aninsertion, a duplication, and an inversion of one or more nucleotidesrelative to a wild-type PMT gene. 13.-17. (canceled)
 18. The tobaccoplant, or part thereof, of any one of the preceding claims claim 1,wherein said tobacco plant is capable of producing a leaf comprising anicotine level selected from the group consisting of less than 0.15%,less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, lessthan 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and lessthan 0.01% dry weight. 19.-21. (canceled)
 22. Cured tobacco materialfrom the tobacco plant of any one of claim 1 to
 20. 23. The curedtobacco material of claim 22, wherein said cured tobacco material ismade by a curing process selected from the group consisting of fluecuring, air curing, fire curing, and sun curing.
 24. The cured tobaccomaterial of claim 22, wherein said cured tobacco material comprisestobacco leaf, and wherein said tobacco leaf exhibits reduced moldinfection as compared to control cured tobacco material from the varietyLA Burley
 21. 25.-27. (canceled)
 28. A tobacco product comprising thecured tobacco material of claim
 22. 29. The tobacco product of claim 28,wherein said tobacco product is selected from the group consisting of acigarette, a cigarillo, a non-ventilated recess filter cigarette, avented recess filter cigarette, a cigar, snuff, pipe tobacco, cigartobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shreddedtobacco, and cut tobacco.
 30. The tobacco product of claim 28, whereinsaid tobacco product is a smokeless tobacco product.
 31. The tobaccoproduct of claim 30, wherein said smokeless tobacco product is selectedfrom the group consisting of loose leaf chewing tobacco, plug chewingtobacco, moist snuff, and nasal snuff.
 32. A reconstituted tobaccocomprising the cured tobacco material of claim 22.